Search

Hydroxycinnamic acids such as p-coumaric acid (CA) are chemically linked to lignin in grassy biomass with fairly labile ester bonds and therefore represent a straightforward opportunity to extract and valorize lignin components. In this work, we investigated the enzymatic conversion of CA extracted from lignocellulose to 4-vinylphenol (4VP) by expressing a microbial phenolic acid decarboxylase in Corynebacterium glutamicum, Escherichia coli, and Bacillus subtilis. The performance of the recombinant strains was evaluated in response to the substrate concentration in rich medium or a lignin liquor and the addition of an organic overlay to perform a continuous product extraction in batch cultures. We found that using undecanol as an overlay enhanced the 4VP titers under high substrate concentrations, while extracting > 97% of the product from the aqueous phase. C. glutamicum showed the highest tolerance to CA and resulted in the accumulation of up to 187 g/L of 4VP from pure CA in the overlay with a 90% yield when using rich media, or 17 g/L of 4VP with a 73% yield from CA extracted from lignin. These results indicate that C. glutamicum is a suitable host for the high-level production of 4VP and that further bioprocess engineering strategies should be explored to optimize the production, extraction, and purification of 4VP from lignin with this organism.

The photosynthetic unicellular alga Chlamydomonas (Chlamydomonas reinhardtii) is a versatile reference for algal biology because of its ease of culture in the laboratory. Genomic and systems biology approaches have previously described transcriptome responses to environmental changes using bulk data, thus representing the average behavior from pools of cells. Here, we apply single-cell RNA sequencing (scRNA-seq) to probe the heterogeneity of Chlamydomonas cell populations under three environments and in two genotypes differing by the presence of a cell wall. First, we determined that RNA can be extracted from single algal cells with or without a cell wall, offering the possibility to sample natural algal communities. Second, scRNA-seq successfully separated single cells into nonoverlapping cell clusters according to their growth conditions. Cells exposed to iron or nitrogen deficiency were easily distinguished despite a shared tendency to arrest photosynthesis and cell division to economize resources. Notably, these groups of cells not only recapitulated known patterns observed with bulk RNA-seq but also revealed their inherent heterogeneity. A substantial source of variation between cells originated from their endogenous diurnal phase, although cultures were grown in constant light. We exploited this result to show that circadian iron responses may be conserved from algae to land plants. We document experimentally that bulk RNA-seq data represent an average of typically hidden heterogeneity in the population.

The unicellular green alga Chlamydomonas reinhardtii is a choice reference system for the study of photosynthesis and chloroplast metabolism, cilium assembly and function, lipid and starch metabolism, and metal homeostasis. Despite decades of research, the functions of thousands of genes remain largely unknown, and new approaches are needed to categorically assign genes to cellular pathways. Growing collections of transcriptome and proteome data now allow a systematic approach based on integrative co-expression analysis. We used a dataset comprising 518 deep transcriptome samples derived from 58 independent experiments to identify potential co-expression relationships between genes. We visualized co-expression potential with the R package corrplot, to easily assess co-expression and anti-correlation between genes. We extracted several hundred high-confidence genes at the intersection of multiple curated lists involved in cilia, cell division, and photosynthesis, illustrating the power of our method. Surprisingly, Chlamydomonas experiments retained a significant rhythmic component across the transcriptome, suggesting an underappreciated variable during sample collection, even in samples collected in constant light. Our results therefore document substantial residual synchronization in batch cultures, contrary to assumptions of asynchrony. We provide step-by-step protocols for the analysis of co-expression across transcriptome data sets from Chlamydomonas and other species to help foster gene function discovery.

BackgroundMagnetic nanoparticles such as magnetosomes modified with antibodies allow a high probability of their interaction with targets of interest. Magnetosomes biomineralized by magnetotactic bacteria are in homogeneous nanoscale size and have crystallographic structure, and high thermal and colloidal stability. Camelidae derived nanobodies (Nbs) are small in size, thermal stable, highly water soluble, easy to produce, and fusible with magnetosomes. We aimed to functionalize Nb-magnetosomes for the analysis of the insecticide fipronil.ResultsThree recombinant magnetotactic bacteria (CF, CF+ , and CFFF) biomineralizing magnetosomes with different abundance of Nbs displayed on the surface were constructed. Compared to magnetosomes from the wild type Magnetospirillum gryphiswaldense MSR-1, all of the Nb-magnetosomes biosynthesized by strains CF, CF+ , and CFFF showed a detectable level of binding capability to fipronil-horseradish peroxidase (H2-HRP), but none of them recognized free fipronil. The Nb-magnetosomes from CFFF were oxidized with H2O2 or a glutathione mixture consisting of reduced glutathione and oxidized glutathione in vitro and their binding affinity to H2-HRP was decreased, whereas that to free fipronil was enhanced. The magnetosomes treated with the glutathione mixture were employed to develop an enzyme-linked immunosorbent assay for the detection of fipronil in water samples, with average recoveries in a range of 78-101%.ConclusionsThe economical and environmental-friendly Nb-magnetosomes biomineralized by the bacterial strain MSR-1 can be potentially applied to nanobody-based immunoassays for the detection of fipronil or nanobody-based assays in general.

High titer, rate, yield (TRY), and scalability are challenging metrics to achieve due to trade-offs between carbon use for growth and production. To achieve these metrics, we take the minimal cut set (MCS) approach that predicts metabolic reactions for elimination to couple metabolite production strongly with growth. We compute MCS solution-sets for a non-native product indigoidine, a sustainable pigment, in Pseudomonas putida KT2440, an emerging industrial microbe. From the 63 solution-sets, our omics guided process identifies one experimentally feasible solution requiring 14 simultaneous reaction interventions. We implement a total of 14 genes knockdowns using multiplex-CRISPRi. MCS-based solution shifts production from stationary to exponential phase. We achieve 25.6 g/L, 0.22 g/l/h, and ~50% maximum theoretical yield (0.33 g indigoidine/g glucose). These phenotypes are maintained from batch to fed-batch mode, and across scales (100-ml shake flasks, 250-ml ambr®, and 2-L bioreactors).

BackgroundThe physiological characterization of microorganisms provides valuable information for bioprocess development. Chemostat cultivations are a powerful tool for this purpose, as they allow defined changes to one single parameter at a time, which is most commonly the growth rate. The subsequent establishment of a steady state then permits constant variables enabling the acquisition of reproducible data sets for comparing microbial performance under different conditions. We performed physiological characterizations of a 3-hydroxypropionic acid (3-HP) producing Saccharomyces cerevisiae strain in a miniaturized and parallelized chemostat cultivation system. The physiological conditions under investigation were various growth rates controlled by different nutrient limitations (C, N, P). Based on the cultivation parameters obtained subsequent fed-batch cultivations were designed.ResultsWe report technical advancements of a small-scale chemostat cultivation system and its applicability for reliable strain screening under different physiological conditions, i.e. varying dilution rates and different substrate limitations (C, N, P). Exploring the performance of an engineered 3-HP producing S. cerevisiae strain under carbon-limiting conditions revealed the highest 3-HP yields per substrate and biomass of 16.6 %C-mol and 0.43 g gCDW-1, respectively, at the lowest set dilution rate of 0.04 h-1. 3-HP production was further optimized by applying N- and P-limiting conditions, which resulted in a further increase in 3-HP yields revealing values of 21.1 %C-mol and 0.50 g gCDW-1 under phosphate-limiting conditions. The corresponding parameters favoring an increased 3-HP production, i.e. dilution rate as well as C- and P-limiting conditions, were transferred from the small-scale chemostat cultivation system to 1-L bench-top fermenters operating in fed-batch conditions, revealing 3-HP yields of 15.9 %C-mol and 0.45 g gCDW-1 under C-limiting, as well as 25.6 %C-mol and 0.50 g gCDW-1 under phosphate-limiting conditions.ConclusionsSmall-scale chemostat cultures are well suited for the physiological characterization of microorganisms, particularly for investigating the effect of changing cultivation parameters on microbial performance. In our study, optimal conditions for 3-HP production comprised (i) a low dilution rate of 0.04 h-1 under carbon-limiting conditions and (ii) the use of phosphate-limiting conditions. Similar 3-HP yields were achieved in chemostat and fed-batch cultures under both C- and P-limiting conditions proving the growth rate as robust parameter for process transfer and thus the small-scale chemostat system as powerful tool for process optimization.

Isoprenol (3-methyl-3-buten-1-ol) is a drop-in biofuel and a precursor for commodity chemicals. Biological production of isoprenol via the mevalonate pathway has been developed and optimized extensively in Escherichia coli, but high ATP requirements and isopentenyl diphosphate (IPP) toxicity have made it difficult to achieve high titer, yield, and large-scale production. To overcome these limitations, an IPP-bypass pathway was previously developed using the promiscuous activity of diphosphomevalonate decarboxylase, and enabled the production of isoprenol at a comparable yield and titer to the original pathway. In this study, we optimized this pathway, substantially improving isoprenol production. A titer of 3.7 g/L (0.14 g isoprenol per g glucose) was achieved in batch conditions using minimal medium by pathway optimization, and a further optimization of the fed-batch fermentation process enabled an isoprenol titer of 10.8 g/L (yield of 0.105 g/g and maximum productivity of 0.157 g L-1 h-1), which is the highest reported titer for this compound. The substantial increase in isoprenol titer via the IPP-bypass pathway in this study will facilitate progress toward commercialization.

Microbial fermentation is an essential process for research and industrial applications, yet our understanding of cellular dynamics during long-term fermentation is limited. Here, we report a reproducible phenomenon of abrupt population collapse followed by a rapid population rescue that was observed during long-term chemostat cultivations, for various strains of Escherichia coli in minimal media. Through genome resequencing and whole-genome transcriptional profiling of replicate runs over time, we identified that changes in the tRNA and carbon catabolic genes are the genetic basis of this phenomenon. Since current fermentation models are unable to capture the observed dynamics, we present an extended model that takes into account critical biological processes during fermentation, and we further validated carbon source predictions through forward experimentation. This study extends the predictability of current models for microbial fermentation and adds to our system-level knowledge of cellular adaptation during this crucial biotechnological process.

Gluconic acid, an oxidized cellulose degradation product, could be produced from cellulosic biomass. Glycerol is an inexpensive and renewable resource for fuels and chemicals production and is available as a byproduct of biodiesel production. Gluconate is a more oxidized substrate than glucose, whereas glycerol is a more reduced substrate than glucose. Although the production of homoethanol from glucose can be achieved, the conversion of gluconate to ethanol is accompanied by the production of oxidized byproduct such as acetate, and reduced byproducts such as 1,3-propanediol are produced, along with ethanol, when glycerol is used as the carbon source. When gluconate and glycerol are used as the sole carbon source by Klebsiella oxytoca BW21, the ethanol yield is about 62 to 64%. Coutilization of both gluconate and glycerol in batch fermentation increased the yield of ethanol to about 78.7% and decreased by-product accumulation (such as acetate and 1,3-propanediol) substantially. Decreasing by-product formation by deleting the pta, frd, ldh, pflA, and pduC genes in strain BW21 increased the ethanol yield to 89.3% in the batch fermentation of a glycerol-gluconate mixture. These deletions produced the strain K. oxytoca WT26. However, the utilization rate of glycerol was significantly slower than that of gluconate in batch fermentation. In addition, substantial amounts of glycerol remain unutilized after gluconate was depleted in batch fermentation. Continuous fed-batch fermentation was used to solve the utilization rate mismatch problem for gluconate and glycerol. An ethanol yield of 97.2% was achieved in continuous fed-batch fermentation of these two substrates, and glycerol was completely used at the end of the fermentation.IMPORTANCE Gluconate is a biomass-derived degradation product, and glycerol can be obtained as a biodiesel byproduct. Compared to glucose, using them as the sole substrate is accompanied by the production of by-products. Our study shows that through pathway engineering and adoption of a fed-batch culture system, high-yield homoethanol production that usually can be achieved by using glucose as the substrate is achievable using gluconate and glycerol as cosubstrates. The same strategy is expected to be able to achieve homofermentative production of other products, such as lactate and 2,3-butanediol, which can be typically achieved using glucose as the substrate and inexpensive biodiesel-derived glycerol and biomass-derived gluconate as the cosubstrates.

With 70% of the Earth's surface covered in water, the marine ecosystem offers immense opportunities for drug discovery and development. Due to the decreasing rate of novel natural product discovery from terrestrial sources in recent years, many researchers are beginning to look seaward for breakthroughs in new therapeutic agents. As part of an ongoing marine drug discovery programme in Singapore, an integrated approach of combining metabolomic and genomic techniques were initiated for uncovering novel anti-quorum sensing molecules from bacteria associated with subtidal samples collected in the Singapore Strait. Based on the culture-dependent method, a total of 102 marine bacteria strains were isolated and the identities of selected strains were established based on their 16S rRNA gene sequences. About 5% of the marine bacterial organic extracts showed quorum sensing inhibitory (QSI) activity in a dose-dependent manner based on the Pseudomonas aeruginosa QS reporter system. In addition, the extracts were subjected to mass spectrometry-based molecular networking and the genome of selected strains were analysed for known as well as new biosynthetic gene clusters. This study revealed that using integrated techniques, coupled with biological assays, can provide an effective and rapid prioritization of marine bacterial strains for downstream large-scale culturing for the purpose of isolation and structural elucidation of novel bioactive compounds.

BackgroundWe previously developed an E. coli strain that overproduces medium-chain methyl ketones for potential use as diesel fuel blending agents or as flavors and fragrances. To date, the strain's performance has been optimized during growth with glucose. However, lignocellulosic biomass hydrolysates also contain a substantial portion of hemicellulose-derived xylose, which is typically the second most abundant sugar after glucose. Commercialization of the methyl ketone-producing technology would benefit from the increased efficiency resulting from simultaneous, rather than the native sequential (diauxic), utilization of glucose and xylose.ResultsIn this study, genetic manipulations were performed to alleviate carbon catabolite repression in our most efficient methyl ketone-producing strain. A strain engineered for constitutive expression of xylF and xylA (involved in xylose transport and metabolism) showed synchronized glucose and xylose consumption rates. However, this newly acquired capability came at the expense of methyl ketone titer, which decreased fivefold. Further efforts were made to improve methyl ketone production in this strain, and we found that two strategies were effective at enhancing methyl ketone titer: (1) chromosomal deletion of pgi (glucose-6-phosphate isomerase) to increase intracellular NADPH supply and (2) downregulation of CRP (cAMP receptor protein) expression by replacement of the native RBS with an RBS chosen based upon mutant library screening results. Combining these strategies resulted in the most favorable overall phenotypes for simultaneous glucose-xylose consumption without compromising methyl ketone titer at both 1 and 2% total sugar concentrations in shake flasks.ConclusionsThis work demonstrated a strategy for engineering simultaneous utilization of C6 and C5 sugars in E. coli without sacrificing production of fatty acid-derived compounds.

This work focused on the physico-chemical, biochemical and microbiological characterization of a new organic fertilizer based on fermented forest litter (FFL) mixed with agro-industrial by-products, on the culture realized in airtight glass bottle. Under strict anaerobiosis (0% initial oxygen concentration (IOC)), after a 16-day batch culture, the bottle-headspace analysis showed that the specific CO 2 production rate was low (0.014 mL/h.g dry matter) compared to those reached under aerobic conditions (e.g. 0.464 mL/h.g dm at 21% IOC). Moreover, the culture displayed a slight fermented fruity odour, mainly due to ethanol and ethyl acetate detected in the headspace (335 µL and 58.6 µL accumulated, respectively). The FFL organic matter degradation followed by infrared spectroscopy and catabolic potential and diversity characterized by BIOLOG ® EcoPlates were poor and pH dropped to 4.54. The microbiome's metabolism was oriented toward lactic fermentation with medium acidification, enrichment in lactic acid bacteria (LAB), depletion in fungi and absence of pathogens. By increasing IOC from 0 to 21%, the respirometric activity, and the catabolic potential and diversity increased. However, some enterobacteria were detected above 5% IOC. Ethanol and ethyl acetate decreased strongly with IOC, and aromatics and proteins contained in the solid matrix remained in the culture. This study showed the importance of oxygen on the final product. A 2% IOC was found to ensure an optimal balance between LAB development, preservation of functional catabolic diversity and bio-product free of microbial pathogens., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Background: With a growing global population, the generation of plastic waste and the depletion of fossil resources are major concerns that need to be addressed by developing sustainable and efficient plastic recycling methods. Biocatalytic recycling is emerging as a promising ecological alternative to conventional processes, particularly in the recycling of polyethylene terephthalate (PET). However, cost-effective production of the involved biocatalyst is essential for the transition of enzymatic PET recycling to a widely used industrial technology. Extracellular enzyme production using established organisms such as Escherichia coli or Corynebacterium glutamicum offers a promising way to reduce downstream processing costs., Results: In this study, we compared extracellular recombinant protein production by classical secretion in C. glutamicum and by membrane leakage in E. coli. A superior extracellular release of the cutinase ICCG DAQI was observed with E. coli in batch and fed-batch processes on a litre-scale. This phenomenon in E. coli, in the absence of a signal peptide, might be associated with membrane-destabilizing catalytic properties of the expressed cutinase. Optimisations regarding induction, expression temperature and duration as well as carbon source significantly enhanced extracellular cutinase activity. In particular, in fed-batch cultivation of E. coli at 30 °C with lactose as carbon source and inducer, a remarkable extracellular activity (137 U mL -1 ) and cutinase titre (660 mg L -1 ) were achieved after 48 h. Literature values obtained with other secretory organisms, such as Bacillus subtilis or Komagataella phaffii were clearly outperformed. The extracellular ICCG DAQI produced showed high efficacy in the hydrolysis of PET textile fibres, either chromatographically purified or unpurified as culture supernatant. In less than 18 h, 10 g L -1 substrate was hydrolysed using supernatant containing 3 mg cutinase ICCG DAQI at 70 °C, pH 9 with terephthalic acid yields of up to 97.8%., Conclusion: Extracellular production can reduce the cost of recombinant proteins by simplifying downstream processing. In the case of the PET-hydrolysing cutinase ICCG DAQI , it was even possible to avoid chromatographic purification and still achieve efficient PET hydrolysis. With such production approaches and their further optimisation, enzymatic recycling of PET can contribute to a more efficient and environmentally friendly solution to the industrial recycling of plastics in the future., (© 2024. The Author(s).)

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.)

Single-cell protein (SCP) derived from microorganisms is widely recognized as a viable alternative protein source for the future. Nevertheless, the commercialization of yeast-based SCP is hampered by its relatively low protein content. Therefore, this study aimed to enhance the protein content of Saccharomyces cerevisiae via random mutagenesis. To achieve this, S. cerevisiae KCCM 51811, which exhibited the highest protein concentration among 20 edible S. cerevisiae strains, was selected as a chassis strain. Subsequently, a KCCM 51811 mutant library was constructed (through UV irradiation) and screened to isolate mutants exhibiting high protein content and/or concentration. Among the 174 mutant strains studied, the #126 mutant exhibited a remarkable 43% and 36% higher protein content and concentration, respectively, compared to the parental strain. Finally, the #126 mutant was cultured in a fed-batch system using molasses and corn-steep liquor, resulting in a protein concentration of 21.6 g/l in 100 h, which was 18% higher than that produced by the parental strain. These findings underscore the potential of our approach for the cost-effective production of food-grade SCP.

Poly-γ-glutamic acid (PGA) has been of interest as a sustainable biopolymer in industrial applications. PGA biosynthesis in Bacillus subtilis is catalyzed by a transmembrane protein complex comprising PgsB, PgsC, and PgsA. To determine the Pgs component responsible for PGA overproduction, we constructed recombinants in which the promoter of the host-derived pgs gene was replaced with another host-derived gene promoter. These recombinants were then transformed using high-copy-number plasmids with various pgs-gene combinations to enhance Pgs component in different ratios. Subsequently, PGA production was investigated in batch cultures with l-glutamate supplemented medium. The recombinant strain enhanced with pgsB alone significantly overproduced PGA (maximum production 35.8 g/L) than either the pgsC- or pgsA-enhanced strain. The molecular weight of the PGA produced with the pgsB-enhanced strain was also greater than that for the pgsC- or pgsA-enhanced strain (approximately 10-fold). Hence, PgsB enhancement alone contributes to PGA overproduction with increased molecular weight., (© The Author(s) 2024. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

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 occurrence of mutations is a cornerstone of the evolutionary theory of adaptation, capitalizing on the rare chance that a mutation confers a fitness benefit. Natural selection is increasingly being leveraged in laboratory settings for industrial and basic science applications. Despite increasing deployment, there are no standardized procedures available for designing and performing adaptive laboratory evolution (ALE) experiments. Thus, there is a need to optimize the experimental design, specifically for determining when to consider an experiment complete and for balancing outcomes with available resources (i.e., laboratory supplies, personnel, and time). To design and to better understand ALE experiments, a simulator, ALEsim, was developed, validated, and applied to the optimization of ALE experiments. The effects of various passage sizes were experimentally determined and subsequently evaluated with ALEsim, to explain differences in experimental outcomes. Furthermore, a beneficial mutation rate of 10-6.9 to 10-8.4 mutations per cell division was derived. A retrospective analysis of ALE experiments revealed that passage sizes typically employed in serial passage batch culture ALE experiments led to inefficient production and fixation of beneficial mutations. ALEsim and the results described here will aid in the design of ALE experiments to fit the exact needs of a project while taking into account the resources required and will lower the barriers to entry for this experimental technique.IMPORTANCE ALE is a widely used scientific technique to increase scientific understanding, as well as to create industrially relevant organisms. The manner in which ALE experiments are conducted is highly manual and uniform, with little optimization for efficiency. Such inefficiencies result in suboptimal experiments that can take multiple months to complete. With the availability of automation and computer simulations, we can now perform these experiments in an optimized fashion and can design experiments to generate greater fitness in an accelerated time frame, thereby pushing the limits of what adaptive laboratory evolution can achieve.

Tissue engineering (TE) approaches that involve seeding cells into predetermined tissue scaffolds ignore the complex environment where the material properties are spatially inhomogeneous and evolve over time. We present a new approach for controlling mechanical forces inside bioreactors, which enables spatiotemporal control of flow fields in real time. Our adaptive approach offers the flexibility of dialing-in arbitrary shear stress distributions and adjusting flow field patterns in a scaffold over time in response to cell growth without needing to alter scaffold structure. This is achieved with a multi-inlet bioreactor and a control algorithm with learning capabilities to dynamically solve the inverse problem of computing the inlet pressure distribution required over the multiple inlets to obtain a target flow field. The new method constitutes a new platform for studies of cellular responses to mechanical forces in complex environments and opens potentially transformative possibilities for TE.

3D printing of biological architectures that mimic the structural and functional features of in vivo tissues is of great interest in tissue engineering and the development of transplantable organ constructs. Printable bio-inks that are compatible with cellular activities play critical roles in the process of 3D bio-printing. Although a variety of hydrogels have been used as bio-inks for 3D bio-printing, they inherit poor mechanical properties and/or the lack of essential protein components that compromise their performance. Here, a hybrid Matrigel-agarose hydrogel system has been demonstrated that possesses both desired rheological properties for bio-printing and biocompatibility for long-term (11 days) cell culture. The agarose component in the hybrid hydrogel system enables the maintenance of 3D-printed structures, whereas Matrigel provides essential microenvironments for cell growth. When human intestinal epithelial HCT116 cells are encapsulated in the printed Matrigel-agarose constructs, high cell viability and proper cell spreading morphology are observed. Given that Matrigel is used extensively for 3D cell culturing, the developed 3D-printable Matrigel-agarose system will open a new way to construct Matrigel-based 3D constructs for cell culture and tissue engineering.

The marine alga Nannochloropsis oceanica can synthesize the high-value ketocarotenoid canthaxanthin yet at an extremely low level. Introducing a β-carotenoid ketolase from Chlamydomonas reinhardtii into the chloroplast for expression, enabled N. oceanica to synthesize substantial amounts of canthaxanthin and grow better under high light. Compared to wild type, the engineered strain had higher levels of primary carotenoids and chlorophyll a as well, and synthesized more eicosapentaenoic acid (EPA, an ω3 polyunsaturated fatty acids). Further metabolic engineering by enhancing the flux to carotenoids or suppressing competing pathways allowed for a considerable increase of canthaxanthin, reaching 4.7 mg g -1 dry weight. A fed-batch culture strategy with nitrate and phosphate replenishment was developed for the co-production of canthaxanthin and EPA, which within a 10-day period reached 37.6 and 268.8 mg/L, respectively. This study sheds light on manipulating the industrially relevant alga for efficient co-production of high-value biochemicals from CO 2 ., 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.)

Glycolipids are a class of widely studied biosurfactants with excellent applicability in cosmetic and pharmaceutical formulations. This class of biosurfactants includes mannosylerythritol lipids (MELs), which have gained particular interest due to their moisturizing and healing activity for dry and damaged human skin, arising from conditions such as eczema. Traditionally, MELs have been produced by growing certain basidiomycetous yeasts on vegetable oils. However, oils are a comparatively expensive substrate, which negatively affects the economic performance of MEL production. In addition to this, vegetable oils significantly complicate the downstream processing required to produce a product with the required purity for most applications. To address these challenges, this study investigated MEL-A production exclusively from hydrophilic carbon sources by Ustilago maydis DSM 4500. By implementing a fed-batch production strategy, maximum MEL-A concentration of 0.87 g/L was achieved from glucose exclusively. Also, adding micronutrients (such as MnSO 4 ) to MEL-A production showed a 24.1% increase in the product titer, implying other metabolites are formed, favoring MEL production., (© 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.)

Nannochloropsis oceanica is an industrially relevant marine microalga rich in eicosapentaenoic acid (EPA, a valuable ω-3 polyunsaturated fatty acid), yet the algal production potential remains to be unlocked. Here we engineered N. oceanica to synthesize the high-value carotenoid astaxanthin independent of high-light (HL) induction for achieving multifaceted benefits. By screening β-carotenoid ketolases and hydroxylases of various origins, and strategically manipulating compartmentalization, fusion patterns, and linkers of the enzyme pair, a remarkable 133-fold increase in astaxanthin content was achieved in N. oceanica. Iterative metabolic engineering efforts led to further increases in astaxanthin synthesis up to 7.3 mg g -1 , the highest reported for microalgae under nonstress conditions. Astaxanthin was found in the photosystem components and allowed the alga HL resistance and augmented EPA production. Besides, we achieved co-production of astaxanthin and EPA by the engineered alga through a fed-batch cultivation approach. Our findings unveil the untapped potential of N. oceanica as a robust, light-driven chassis for constitutive astaxanthin synthesis and provide feasible strategies for the concurrent production of multiple high-value biochemicals from CO 2 , thereby paving the way for sustainable biotechnological applications of this alga., (© 2024 The Author(s). New Phytologist © 2024 New Phytologist Foundation.)

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.)

The enzymatic hydrolysis of cellulosic biomass is a key step in the biochemical production of fuels and chemicals. Economically feasible large-scale implementation of the process requires operation at high solids loadings, i.e., biomass concentrations >15% (w/w). At increasing solids loadings, however, biomass forms a high viscosity slurry that becomes increasingly challenging to mix and severely mass transfer limited, which limits further addition of solids. To overcome these limitations, we developed a fed-batch process controlled by the yield stress and its changes during liquefaction of the reaction mixture. The process control relies on an in-line, non-invasive magnetic resonance imaging (MRI) rheometer to monitor real-time evolution of yield stress during liquefaction. Additionally, we demonstrate that timing of enzyme addition relative to biomass addition influences process efficiency, and the upper limit of solids loading is ultimately limited by end-product inhibition as soluble glucose and cellobiose accumulate in the liquid phase.

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).)

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.)

Development of stem cell delivery system with ability of control over mutilineage differentiation and improved engraft efficiency is imperative in regenerative medicine. We herein report transfer stamping of human mesenchymal stem cells (hMSCs) patches using thermally expandable hydrogels with tunable cell-adhesive properties. The hydrogels were prepared from functionalized four arm copolymer of Tetronic(®), and the cell adhesion on the hydrogel was modulated by incorporation of fibronectin (FN) or cell-adhesive peptide (RGD). The resulting hydrogels showed spontaneous expansion in size within 10 min in response to the temperature reduction from 37 to 4°C. The adhesion and proliferation of hMSCs on FN-hydrogels were positively tunable in proportion to the amount of FN within hydrogels with complete monolayer of hMSCs (hMSC patch) being successfully achieved. The hMSC patch on the hydrogel was faced to the target substrate, which was then easily detached and re-attached to the target when the temperature was reduced from 37°C up to 4°C. We found that the transfer stamping of cell patch was facilitated at lower temperature of 4°C relative to 25°C, with the use of thinner hydrogels (0.5 mm in thickness relatively to 1.0 or 1.5 mm) and longer transfer time (>15 min). Notably, the hMSC patch was simply transferred from the hydrogel to the subcutaneous mouse skin tissue within 15 min with cold saline solution being dropped to the hydrogel. The hMSC patch following osteogenic or adipogenic commitment was also achieved with long-term culture of hMSCs on the hydrogel, which was successfully detached to the target surface. These results suggest that the hydrogels with thermally expandable and tunable cell-adhesive properties may serve as a universal substrate to harvest hMSC patch in a reliable and effective manner, which could potentially be utilized in many cell-sheet based therapeutic applications.

Human pluripotent stem cells (hPSC) are used to study the early stages of human development in vitro and, increasingly due to somatic cell reprogramming, cellular and molecular mechanisms of disease. Cell culture medium is a critical factor for hPSC to maintain pluripotency and self-renewal. Numerous defined culture media have been empirically developed but never systematically optimized for culturing hPSC. We applied design of experiments (DOE), a powerful statistical tool, to improve the medium formulation for hPSC. Using pluripotency and cell growth as read-outs, we determined the optimal concentration of both basic fibroblast growth factor (bFGF) and neuregulin-1 beta 1 (NRG1β1). The resulting formulation, named iDEAL, improved the maintenance and passage of hPSC in both normal and stressful conditions, and affected trimethylated histone 3 lysine 27 (H3K27me3) epigenetic status after genetic reprogramming. It also enhances efficient hPSC plating as single cells. Altogether, iDEAL potentially allows scalable and controllable hPSC culture routine in translational research. Our DOE strategy could also be applied to hPSC differentiation protocols, which often require numerous and complex cell culture media.

Biobased chemicals have become attractive replacements for their fossil-fuel counterparts. Recent studies have shown triacetic acid lactone (TAL) to be a promising candidate, capable of undergoing chemical conversion to sorbic acid and other valuable intermediates. In this study, Saccharomyces cerevisiae was engineered for the high-level production of TAL by overexpression of the Gerbera hybrida 2-pyrone synthase (2-PS) and systematic engineering of the yeast metabolic pathways. Pathway analysis and a computational approach were employed to target increases in cofactor and precursor pools to improve TAL synthesis. The pathways engineered include those for energy storage and generation, pentose biosynthesis, gluconeogenesis, lipid biosynthesis and regulation, cofactor transport, and fermentative capacity. Seventeen genes were selected for disruption and independently screened for their effect on TAL production; combinations of knockouts were then evaluated. A combination of the pathway engineering and optimal culture parameters led to a 37-fold increase in titer to 2.2g/L and a 50-fold increase in yield to 0.13 (g/g glucose). These values are the highest reported in the literature, and provide a 3-fold improvement in yield over previous reports using S. cerevisiae. Identification of these metabolic bottlenecks provides a strategy for overproduction of other acetyl-CoA-dependent products in yeast.

BackgroundRecent years have witnessed a rising trend in exploring microalgae for valuable carotenoid products as the demand for lutein and many other carotenoids in global markets has increased significantly. In green microalgae lutein is a major carotenoid protecting cellular components from damage incurred by reactive oxygen species under stress conditions. In this study, we investigated the effects of abiotic stressors on lutein accumulation in a strain of the marine microalga D. salina which had been selected for growth under stress conditions of combined blue and red lights by adaptive laboratory evolution.ResultsNitrate concentration, salinity and light quality were selected as three representative influencing factors and their impact on lutein production in batch cultures of D. salina was evaluated using response surface analysis. D. salina was found to be more tolerant to hyper-osmotic stress than to hypo-osmotic stress which caused serious cell damage and death in a high proportion of cells while hyper-osmotic stress increased the average cell size of D. salina only slightly. Two models were developed to explain how lutein productivity depends on the stress factors and for predicting the optimal conditions for lutein productivity. Among the three stress variables for lutein production, stronger interactions were found between nitrate concentration and salinity than between light quality and the other two. The predicted optimal conditions for lutein production were close to the original conditions used for adaptive evolution of D. salina. This suggests that the conditions imposed during adaptive evolution may have selected for the growth optima arrived at.ConclusionsThis study shows that systematic evaluation of the relationship between abiotic environmental stresses and lutein biosynthesis can help to decipher the key parameters in obtaining high levels of lutein productivity in D. salina. This study may benefit future stress-driven adaptive laboratory evolution experiments and a strategy of applying stress in a step-wise manner can be suggested for a rational design of experiments.

The vast majority of nanotoxicity studies measures the effect of exposure to a toxicant on an organism and ignores the potentially important effects of the organism on the toxicant. We investigated the effect of citrate-coated silver nanoparticles (AgNPs) on populations of the freshwater alga Chlamydomonas reinhardtii at different phases of batch culture growth and show that the AgNPs are most toxic to cultures in the early phases of growth. We offer strong evidence that reduced toxicity occurs because extracellular dissolved organic carbon (DOC) compounds produced by the algal cells themselves mitigate the toxicity of AgNPs. We analyzed this feedback with a dynamic model incorporating algal growth, nanoparticle dissolution, bioaccumulation of silver, DOC production and DOC-mediated inactivation of nanoparticles and ionic silver. Our findings demonstrate how the feedback between aquatic organisms and their environment may impact the toxicity and ecological effects of engineered nanoparticles.

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.)