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Chinese hamster ovary (CHO) cells are widely used for constructing expression systems to produce therapeutic proteins. However, the establishment of high-producer clones remains a laborious and time-consuming process, despite various progresses having been made in cell line development. We previously developed a new strategy for screening high monoclonal antibody (mAb)-producing cells using flow cytometry (FCM). We also reported that p180 and SF3b4 play key roles in active translation on the endoplasmic reticulum, and that the productivity of secreted alkaline phosphatase was enhanced by the overexpression of p180 and SF3b4. Here, we attempted to apply the translational enhancing technology to high mAb-producing cells obtained after high-producer cell sorting. A high mAb-producing CHO clone, L003, which showed an mAb production level of3 g/L in fed-batch culture, was established from a high mAb-producing cell pool fractionated by FCM. Clones generated by the overexpression of p180 and SF3b4 in L003 cells were evaluated by fed-batch culture. The specific productivity of clones overexpressing these two factors was ∼3.1-fold higher than that of parental L003 cells in the early phase of the culture period. Furthermore, the final mAb concentration was increased to 9.5 g/L during 17 days of fed-batch culture after optimizing the medium and culture process. These results indicate that the overexpression of p180 and SF3b4 would be promising for establishing high-producer cell lines applicable to industrial production.

Filamentous fungi are used as production hosts for various commercially valuable enzymes and chemicals including organic acids and secondary metabolites. We previously revealed that α-1,3-glucan and galactosaminogalactan (GAG) contribute to hyphal aggregation in the industrial fungus Aspergillus oryzae, and that production of recombinant protein in shake-flask culture is higher in a mutant lacking both α-1,3-glucan and GAG (AGΔ-GAGΔ) than in the parental strain. Here, we compared the productivity of the wild type, AGΔ-GAGΔ, and mutants lacking α-1,3-glucan (AGΔ) or GAG (GAGΔ) in batch culture with intermittent addition of glucose in a 5-L lab-scale bioreactor. The hyphae of the wild type and all mutants were dispersed by agitation, although the wild type and AGΔ formed small amounts of aggregates. Although mycelial weight was similar among the strains, the concentration of a secreted recombinant protein (CutL1) was the highest in AGΔ-GAGΔ. Evaluation of fluid properties revealed that the apparent viscosities of mycelial cultures of the wild type and AGΔ-GAGΔ decreased as the agitation speed was increased. The apparent viscosity of the AGΔ-GAGΔ culture tended to be lower than that of the wild-type strain at each agitation speed, and was significantly lower at 600 rpm. Overall, the lack of α-1,3-glucan and GAG in the hyphae improved culture rheology, resulting in an increase in recombinant protein production in AGΔ-GAGΔ. This is the first report of flow behavior improvement by a cell-surface component defect in a filamentous fungus.

The biopharmaceutical industry must guarantee the efficiency and biosafety of biological medicines, which are quite sensitive to cell culture process variability. Real time monitoring procedures based on vibrational spectroscopy such as NIR spectroscopy, are then emerging to support innovative strategies for retro-control of key parameters as substrates and by-product concentration. Whereas monitoring models are mainly constructed using Partial Least Squares Regression (PLSR), spectroscopic models based on Artificial Neural Networks (ANNR) and Support Vector Regression (SVR) are emerging with promising results. Unfortunately, analysis of their performance in cell culture monitoring has been limited. This work was then focused to assess their performance and suitability for the cell culture process challenges. PLSR had inferior values of the determination coefficient (R2 ) for all the monitored parameters (i.e., 0.85, 0.93, 0.98 respectively for the PLSR, SVR, and ANNR models for glucose). In general, PLSR had a limited performance while models based on ANNR and SVR have been shown superior due to better management of inter-batch heterogeneity and enhanced specificity. Overall, the use of SVR and ANNR for the generation of calibration models enhanced the potential of NIR spectroscopy as a monitoring tool. This article is protected by copyright. All rights reserved.

In the field of therapeutic antibody production, diversification of fed-batch strategies is flourishing in response to the market demand. All manufacturing approaches tend to follow the generally accepted dogma of increasing titer since it directly increases manufacturing output. While titer is influenced by the biomass (expressed as IVCD), the culture time and the cell-specific productivity (qP), we changed independently each of these parameters to tune our process strategy towards adapted solutions to individual manufacturing needs. To do so, we worked separately on the increase of the IVCD as high seeding fed-batch capacity. Yet, as intensified fed-batch may not always be possible due to limited facility operational mode, we also separately increased the qP with the addition of specific media additives. Both strategies improved titer by 100% in 14 days relative to the standard fed-batch process with moderate and acceptable changes in product quality attributes. Since intensified fed-batch could rival the cell-specific productivity of a conventional fed-batch, we developed novel hybrid strategies to either allow for acceptable seeding densities without compromising productivity, or alternatively, to push the productivity the furthest in order to reduce timelines.

Gut microbial fermentation of soluble dietary fibers promotes general and substrate-specific health benefits. In this study, the fermentation characteristics of two soluble branched-dietary fibers, namely, agavin (a type of agave fructans) and digestion-resistant maltodextrin (RD) were investigated against cellulose, using a simulated colonic fermenter apparatus employing a mixed culture of swine fecal bacteria. After 48 h of complete fermentation period, the microbial composition was different among all groups, where Bifidobacterium spp. and Lactobacillus spp. dominated the agavin treatment, while the members of the families Lachnospiraceae and Prevotellaceae dominated the RD treatment. Agavin treatment exhibited a clearly segregated two-phased prolonged fermentation trend compared to RD treatment as manifested by the fermentation rates. Further, the highest short-chain fatty acids production even at the end of the fermentation cycle, acidic pH, and the negligible concentration of ammonia accumulation demonstrated favorable fermentation attributes of agavin compared to RD. Therefore, agavin might be an effective and desirable substrate for the colonic microbiota than RD with reference to the expressed microbial taxa and fermentation attributes. This study revealed a notable significance of the structural differences of fermentable fibers on the subsequent fermentation characteristics.

The initial growth rate of a yeast strain is a key parameter in the production of fermented beverages. Fast growth is linked with higher fermentative capacity and results in less slow and stuck fermentations unable to reach the expected final gravity. As concentrations of metabolites are in a constant state of flux, quantitative data on how growth rate affects the production of aromatic compounds becomes an important factor for brewers. Chemostats allow to set and keep a specific dilution rate throughout the fermentation and are ideal system to study the effect of growth on aroma production. In this study, we ran chemostats alongside batch and fed-batch cultures, compared volatile profiles detected at different growth rates, and identified those affected by the different feeding profiles. Specifically, we quantified six abundant aroma compounds produced in anaerobic glucose-limited continuous cultivations of S. cerevisiae at different dilution rates. We found that volatile production was affected by the growth rate in four out of six compounds assayed, with higher alcohols and esters following opposite trends. Batch and fed-batch fermentations were devised to study the extent by which the final concentration of volatile compounds is influenced by glucose availability. Compared with the batch system, fed-batch fermentations, where the yeast growth was artificially limited by a slow constant release of nutrients in the media, resulted in a significant increase in concentration of higher alcohols, mirroring the results obtained in continuous fermentations. This study paves the way to further process development optimization for the production of fermented beverages.

Biofilm dispersion can be triggered by the application of dispersing agents such as nitric oxide (NO)-donors, resulting in the release of biofilm-dispersed cells into the environment. In this work, biofilm-dispersed cells were obtained by adding different concentrations of NO-donor sodium nitroprusside (0.5, 5, 50 µM, and 2.5 mM of SNP) to batch cultures of pre-formed

Collection of CD3+ lymphocytes via lymphapheresis is essential for manufacturing autologous chimeric antigen receptor (CAR) T cells. Optimization of timing and procedures for lymphapheresis for each patient is critical because patients often have progressive diseases and receive medications that could reduce T cell counts. We conducted a retrospective study of clinical data from 28 patients who underwent lymphapheresis for CD19-directed CAR-T therapy with tisagenlecleucel to identify factors that could affect CD3+ lymphocyte yields. The numbers of CD3+ cells in peripheral blood were significantly correlated with CD3+ cell yields (correlation coefficient r = 0.84), which enabled us to estimate the volume of blood to process before apheresis. We also found that small cell ratio (SCR) at the apheresis site precisely reflected the proportion of lymphocytes, especially in patients without circulating blasts (coefficient of determination: r2 = 0.9). We were able to predict the CD3+ cell yield and prevent excessive apheresis by measuring pre-apheresis circulating CD3+ cell counts and monitoring SCR. Collectively, these results will help us to establish a strategy for optimization of lymphapheresis procedures for CAR-T cell production on a patient-by-patient basis.

We describe a systematic approach to establish predictive models of CHO cell growth, cell metabolism and monoclonal antibody (mAb) formation during biopharmaceutical production. The prediction is based on a combination of an empirical metabolic model connecting extracellular metabolic fluxes with cellular growth and product formation with mixed Monod-inhibition type kinetics that we generalized to every possible external metabolite. We describe the maximum specific growth rate as a function of the integral viable cell density (IVCD). Moreover, we also take into account the accumulation of metabolites in intracellular pools that can influence cell growth. This is possible even without identification and quantification of these metabolites as illustrated with fed-batch cultures of Chinese Hamster Ovary (CHO) cells producing a mAb. The impact of cysteine and tryptophan on cell growth and cell productivity was assessed, and the resulting macroscopic model was successfully used to predict the impact of new, untested feeding strategies on cell growth and mAb production. This model combining piecewise linear relationships between metabolic rates, growth rate and production rate together with Monod-inhibition type models for cell growth did well in predicting cell culture performance in fed-batch cultures even outside the range of experimental data used for establishing the model. It could therefore also successfully be applied for in silico prediction of optimal operating conditions.

Background The human Bri2 BRICHOS domain inhibits amyloid formation and toxicity and could be used as a therapeutic agent against amyloid diseases. For translation into clinical use, large quantities of correctly folded recombinant human (rh) Bri2 BRICHOS are required. To increase the expression and solubility levels of rh Bri2 BRICHOS it was fused to NT*, a solubility tag derived from the N-terminal domain of a spider silk protein, which significantly increases expression levels and solubility of target proteins. To increase the expression levels even further and reach the g/L range, which is a prerequisite for an economical production on an industrial scale, we developed a fed-batch expression protocol for Escherichia coli. Results A fed-batch production method for NT*-Bri2 BRICHOS was set up and systematically optimized. This gradual improvement resulted in expression levels of up to 18.8 g/L. Following expression, NT*-Bri2 BRICHOS was purified by chromatographic methods to a final yield of up to 6.5 g/L. After removal of the NT*-tag and separation into different oligomeric species, activity assays verified that different assembly states of the fed-batch produced rh Bri2 BRICHOS have the same ability to inhibit fibrillar and non-fibrillar protein aggregation as the reference protein isolated from shake flask cultures. Conclusions The protocol developed in this work allows the production of large quantities of rh Bri2 BRICHOS using the solubility enhancing NT*-tag as a fusion partner, which is required to effectively conduct pre-clinical research.

Since the approval of recombinant human plasminogen activator in 1987, the first protein commercially expressed in Chinese hamster ovary (CHO) cells, there have been tremendous improvements in biopharmaceutical production concerning cell line development, cell culture media and cultivation techniques resulting in productivities exceeding 10 g/L. Today, cell line development is not only focusing on productivity levels but also on product quality. Especially the N-glycosylation pattern is important as it determines pharmacokinetics and pharmacodynamics of a recombinant product and plays a pivotal role for the development of biosimilars. Optimization of cell line performance can be achieved by various approaches, e.g. host cell line or expression vector modifications. The latter includes for example stronger promoters, chromatin opening elements, mRNA stabilizing elements or signal peptides. In recent years, also miRNAs have been applied as tool to improve the productivity of cell lines, at least for difficult-to-express proteins. MiRNAs are small, about 22 nucleotides long RNAs that regulate gene expression. A specific miRNA can bind several target mRNAs leading to translational repression or mRNA degradation. The present study investigated whether miRNAs can be used as a tool to further increase productivities of high-expressing cell lines and to optimize the glycosylation pattern of the recombinant protein. For that purpose, next generation sequencing of small RNAs was performed on 24 CHO DG44 cell lines producing recombinant antibodies. The resulting miRNA expression patterns were subjected to bioinformatic analyses in order to find correlations with high productivity as well as low galactosylation, fucosylation or mannosylation of the antibody. In that way, 30 interesting miRNAs were identified and stably overexpressed in antibody producing CHO DG44 cell lines to verify a potential impact on cell performance. However, none of these miRNAs seemed to have an effect on productivity, growth or the glycosylation pattern. However, further transient testing using miRNA mimics revealed five miRNAs - miR-30c, miR-30e, miR-16 and two novel miRNAs - that reduced galactosylation significantly from 40 % to 36 %. In a further set of experiments, it could be demonstrated that miRNA function seems to be dependent on the cultivation mode, as miR-1 mediated downregulation of TWF1 mRNA was visible in transient and stable batch expression but not during fed-batch cultivation. In conclusion, the overexpression of some specific miRNAs was shown to influence the glycosylation pattern of recombinant CHO DG44 producer cells. However, the observed effects are marginal and thus of limited relevance for cell line engineering, especially as an impact could only be detected on transient level. The identified correlations between miRNA expression and productivity or distinct glycosylation patterns did not have a cause-effect relationship for most miRNAs, highlighting the importance of biological testing upon expression pattern and correlation analyses. Interestingly, it was observed that miRNA function seems to be dependent on the cultivation mode. This is of utmost importance when working on miRNA function evaluation with the goal to apply miRNAs as engimiRs, as most miRNA function studies rely on transient testing in batch mode. However, the present data imply, that miRNAs have to be tested in the cultivation environment that is used in the final application, e.g. fed-batch bioprocess with a stably expressing cell clone for biopharmaceutical production.

Background Fed-batch mode is the standard culture technology for industrial bioprocesses. Nevertheless, most of the early-stage cell and process development is carried out in batch cultures, which can bias the initial selection of expression systems. Cell engineering can provide an alternative to fed-batch cultures for high-throughput screening and host selection. We have previously reported a library of Escherichia coli strains with single and multiple deletions of genes involved in glucose transport. Compared to their wild type (W3110), the mutant strains displayed lower glucose uptake, growth and aerobic acetate production rates. Therefore, when cultured in batch mode, such mutants may perform similar to W3110 cultured in fed-batch mode. To test that hypothesis, we evaluated the constitutive expression of the green fluorescence protein (GFP) in batch cultures in microbioreactors using a semi defined medium supplemented with 10 or 20 g/L glucose + 0.4 g yeast extract/g glucose. Results The mutant strains cultured in batch mode displayed a fast-growth phase (growth rate between 0.40 and 0.60 h−1) followed by a slow-growth phase (growth rate between 0.05 and 0.15 h−1), similar to typical fed-batch cultures. The phase of slow growth is most probably caused by depletion of key amino acids. Three mutants attained the highest GFP fluorescence. Particularly, a mutant named WHIC (ΔptsHIcrr, ΔmglABC), reached a GFP fluorescence up to 14-fold greater than that of W3110. Strain WHIC was cultured in 2 L bioreactors in batch mode with 100 g/L glucose + 50 g/L yeast extract. These cultures were compared with exponentially fed-batch cultures of W3110 maintaining the same slow-growth of WHIC (0.05 h−1) and using the same total amount of glucose and yeast extract than in WHIC cultures. The WHIC strain produced approx. 450 mg/L GFP, while W3110 only 220 mg/L. Conclusion The combination of cell engineering and high throughput screening allowed the selection of a particular mutant that mimics fed-batch behavior in batch cultures. Moreover, the amount of GFP produced by the strain WHIC was substantially higher than that of W3110 under both, batch and fed-batch schemes. Therefore, our results represent a valuable technology for accelerated bioprocess development.

Chondroitin sulfate (CS) is a linear polysaccharide, which is widely used in medical, health care and other fields. Compared with the traditional animal tissue extraction method, microbial synthesis of CS has the advantages of controllability and easiness of scaling-up. In order to achieve an efficient synthesis of chondroitin sulfate A (CSA), we constructed a recombinant

Heparosan is used as the starting polysaccharide sulfated using sulfotransferase to generate fully elaborate heparin, a widely used clinical drug. However, the preparation of heparosan and enzymes was considered tedious since such material must be prepared in separate fermentation batches. In this study, a commonly admitted probiotic, Escherichia coli strain Nissle 1917 (EcN), was engineered to intracellularly express sulfotransferases and, simultaneously, secreting heparosan into the culture medium. The engineered strain EcN::T7M, carrying the λDE3 region of BL21(DE3) encoding T7 RNA polymerase, expressed the sulfotransferase domain (NST) of human N-deacetylase/N-sulfotransferase-1 (NDST-1) and the catalytic domain of mouse 3-O-sulfotransferase-1 (3-OST-1) in a flask. The fed-batch fermentation of EcN::T7M carrying the plasmid expressing NST was carried out, which brought the yield of NST to 0.21 g/L and the yield of heparosan to 0.85 g/L, respectively. Furthermore, the heparosan was purified, characterized by 1H nuclear magnetic resonance (NMR), and sulfated by NST using 3′-phosphoadenosine-5′-phosphosulfate (PAPS) as the sulfo donor. The analysis of element composition showed that over 80% of disaccharide repeats of heparosan were N-sulfated. These results indicate that EcN::T7M is capable of preparing sulfotransferase and heparosan at the same time. The EcN::T7M strain is also a suitable host for expressing exogenous proteins driven by tac promoter and T7 promoter.

Commercial production of therapeutic proteins using mammalian cells requires complex process solutions, and consistency of these process solutions is critical to maintaining product titer and quality between batches. Inconsistencies between process solutions prepared at bench and commercial scale may be due to differences in mixing time, temperature, and pH which can lead to precipitation and subsequent removal via filtration of critical solution components such as trace metals. Pourbaix diagrams provide a useful tool to model the solubility of trace metals and were applied to troubleshoot the scale-up of nutrient feed preparation after inconsistencies in product titer were observed between bench- and manufacturing-scale batches. Pourbaix diagrams modeled the solubility of key metals in solution at various stages of the nutrient feed preparation and identified copper precipitation as the likely root cause of inconsistent medium stability at commercial scale. Copper precipitation increased proportionally with temperature in bench-scale preparations of nutrient feed and temperature was identified as the root cause of copper precipitation at the commercial scale. Additionally, cell culture copper titration studies performed in bench-scale bioreactors linked copper-deficient mammalian cell culture to inconsistent titers at the commercial scale. Pourbaix diagrams can predict when trace metals are at risk of precipitating and can be used to mitigate risk during the scale-up of complex medium preparations.

Riboflavin is widely regarded as an essential nutrient that is involved in biological oxidation in vivo. In addition to preventing and treating acyl-CoA dehydrogenase deficiency in patients with keratitis, stomatitis, and glossitis, riboflavin is also closely related to the treatment of radiation mucositis and cardiovascular disease. Chemical synthesis has been the dominant method for producing riboflavin for approximately 50 years. Nevertheless, due to the intricate synthesis process, relatively high cost, and high risk of pollution, alternative methods of chemical syntheses, such as the fermentation method, began to develop and eventually became the main methods for producing riboflavin. At present, there are three types of strains used in industrial riboflavin production: Ashbya gossypii, Candida famata, and Bacillus subtilis. Additionally, many recent studies have been conducted on Escherichia coli and Lactobacillus. Fermentation increases the yield of riboflavin using genetic engineering technology to modify and induce riboflavin production in the strain, as well as to regulate the metabolic flux of the purine pathway and pentose phosphate pathway (PP pathway), thereby optimizing the culture process. This article briefly introduces recent progress in the fermentation of riboflavin.

Dynamic metabolic engineering that harnesses synthetic biological tools is a next-generation strategy for microbial chemical and fuel production. We previously reported a synthetic quorum sensing system combined with a metabolic toggle switch (QS-MTS) in E. coli. It autonomously redirected endogenous metabolic flux toward the synthetic metabolic pathway and improved biofuel production. However, its functions and effects on host metabolism were attenuated by induction timing delay. Here, we redesigned the QS-MTS to stabilize QS signaling efficiency and metabolic regulation. We performed a metabolome analysis to clarify the effects of QS-MTS redesign on host metabolism. We compared the contributions of conventional and redesigned QS-MTS to fed-batch fermentation. The redesigned QS-MTS was more conducive than the conventional QS-MTS to long-term processes such as fed-batch fermentation. Here, we present a circuit redesign for metabolic flux control based on dynamic characteristic evaluation and metabolome analysis.

Three experiments assessed branched-chain volatile fatty acid (BCVFA) stimulation of neutral detergent fiber (NDF) disappearance after 24 h of incubation in batch cultures derived from ruminal fluid inocula that were enriched with particulate-phase bacteria. In experiment 1, a control was compared with 3 treatments with isomolar doses of all 3 BCVFA (plus valerate), all 3 branched-chain AA (BCAA), or half of each BCVFA and BCAA mix with either alfalfa or grass hays (50%) and ground corn grain (50%). A portion of the BCAA and BCVFA doses were enriched with 13C, and valerate (also enriched with 13C) was added with BCVFA. Although BCAA yielded a similar production of BCVFA compared with dosing BCVFA, equimolar substitution of BCVFA for BCAA decreased the percentage of N in bacterial pellets when alfalfa hay was fed but increased N when grass hay was fed. Substituting BCVFA for BCAA increased total fatty acid (FA) concentration with alfalfa hay. Dosing of BCAA or BCVFA did not affect total branched-chain FA, iso-FA, or anteiso-FA percentages in bacterial total FA, whereas numerous individual FA isomers and their 13C enrichments were affected by these treatments. Increasing recovery of the 13C dose from respective labeled BCVFA primers indicated facilitated BCVFA uptake and incorporation into FA compared with BCAA, whereas increased recovery of 13C from labeled BCAA in the bacteria pellet but not in the FA fraction suggested direct assimilation into bacterial protein. The BCVFA and valerate were dosed in varying combinations that either summed to 4 mM (experiment 2) or had only 1 mM no matter what combination (experiment 3). In general, grass hay was more responsive to stimulation in NDF digestibility by BCVFA than was alfalfa hay, which was attributed to the higher degradable protein in the latter. The net production of the BCVFA (after subtracting dose) was affected by source and combination of BCVFA. Isovalerate dosing tended to increase its own net production; in contrast, isobutyrate seemed to be used more when it was added alone, but 2-methylbutyrate seemed to be preferred over isobutyrate when 2-methylbutyrate was added. Results supported potential interactions, including potential feedback in production from feed BCAA or increased concentration-dependent competition for dosed BCVFA into cellular products. Under our conditions, the BCVFA appear to be more readily available than BCAA, probably because of regulated BCAA transport and metabolism. Valerate consistently provided no benefit. Using nonparametric ranking, all 3 BCVFA or either isovalerate or isobutyrate (both yielding iso-FA) should be combined with 2-methylbutyrate (yielding anteiso-FA) as a potential opportunity to improve NDF digestibility when rumen-degraded BCAA are limited in diets to decrease environmental impact from N in waste.

Streptokinase is a well-established cost-effective therapeutic molecule for thrombo-embolic complications. In the current study, a tag-free variant of streptokinase with a native N-terminus (N-rSK) was developed using the Pichia expression system. A three-copy clone was screened that secreted 1062 mg/L of N-rSK in the complex medium at shake flask level. The biologically active (67,552.61 IU/mg) N-rSK recovered by anion exchange chromatography was predicted to contain 15.43% α-helices, 26.43% β-sheets. The fermentation run in a complex medium yielded a poor quality product due to excessive N-rSK degradation. Therefore, modified basal salt medium was also employed during fermentation operations to reduce the proteolytic processing of the recombinant product. The concomitant feeding of 1 g/L/h soya flour hydrolysate with methanol during the protein synthesis phase reduced the proteolysis and yielded 2.29 g/L of N-rSK. The fermentation medium was also supplemented with urea during growth and induction phases. The combined feeding approach of nitrogen-rich soya flour hydrolysate and urea during bioreactor operations showed significant improvement in protein stability and resulted in a 4-fold increase in N-rSK concentration to a level of 4.03 g/L over shake flask. Under optimized conditions, the volumetric productivity and specific product yield were 52.33 mg/L/h and 33.24 mg/g DCW, respectively.

This study was conducted to enhance the production of tacrolimus in Streptomyces tsukubaensis by strain mutagenesis and optimization of the fermentation medium. A high tacrolimus producing strain S. tsukubaensis FIM-16-06 was obtained by ultraviolet mutagenesis coupled with atmospheric and room temperature plasma mutagenesis.Then, nine variables were screened using Plackett–Burman experimental design, in which soluble starch, peptone and Tween 80 showed significantly affected tacrolimus production. Further studies were carried out employing central composite design to elucidate the mutual interaction between the variables and to work out optimal fermentation medium composition for tacrolimus production. The optimum fermentation medium was found to contain 61.61 g/L of soluble starch, 20.61 g/L of peptone and 30.79 g/L of Tween 80. In the optimized medium, the production of tacrolimus reached 1293 mg/L in shake-flask culture, and reached 1522 mg/L while the scaled-up fermentation was conducted in a 1000 L fermenter, which was about 3.7 times higher than that in the original medium. Combining compound mutation with rational medium optimization is an effective approach for improving tacrolimus production, and the optimized fermentation medium could be efficiently used for industrial production.

Marine actinomycetes from the genus Salinispora have an unexploited biotechnological potential. To accurately estimate their application potential however, data on their cultivation, including biomass growth kinetics, are needed but only incomplete information is currently available. This work provides some insight into the effect of temperature, salinity, nitrogen source, glucose concentration and oxygen supply on growth rate, biomass productivity and yield of Salinispora tropica CBN-440T. The experiments were carried out in unbaffled shake flasks and agitated laboratory-scale bioreactors. The results show that the optimum growth temperature lies within the range 28–30 °C, salinity is close to sea water and the initial glucose concentration is around 10 g/L. Among tested nitrogen sources, yeast extract and soy peptone proved to be the most suitable. The change from unbaffled to baffled flasks increased the volumetric oxygen transfer coefficient (kLa) as did the use of agitated bioreactors. The highest specific growth rate (0.0986 h−1) and biomass productivity (1.11 g/L/day) were obtained at kLa = 28.3 h−1. A further increase in kLa was achieved by increasing stirrer speed, but this led to a deterioration in kinetic parameters. Improvement of S. tropica biomass growth kinetics of was achieved mainly by identifying the most suitable nitrogen sources and optimizing kLa in baffled flasks and agitated bioreactors.

An 8 ton per year manufacturing facility is described based on the framework for integrated and continuous bioprocessing (ICB) common to all known biopharmaceutical implementations. While the output of this plant rivals some of the largest fed‐batch plants in the world, the equipment inside the plant is relatively small: the plant consists of four 2000 L single‐use bioreactors and has a maximum flow rate of 13 L/min. The equipment and facility for the ICB framework is described in sufficient detail to allow biopharmaceutical companies, vendors, contract manufacturers to build or buy their own systems. The design will allow the creation of a global ICB ecosystem that will transform biopharmaceutical manufacturing. The design is fully backward compatible with legacy fed‐batch processes. A clinical production scale is described that can produce smaller batch sizes with the same equipment as that used at the commercial scale. The design described allows the production of as little as 10 g to nearly 35 kg of drug substance per day., An 8 ton per year manufacturing facility is described based on the framework for integrated and continuous bioprocessing (ICB) common to all known biopharmaceutical implementations. The equipment and facility for the ICB framework is described in sufficient detail to allow biopharmaceutical companies, vendors, contract manufacturers to build or buy their own systems. The design will allow the creation of a global ICB ecosystem that will transform biopharmaceutical manufacturing.

Terpenoids have an impressive structural diversity and provide valuable substances for a variety of industrial applications. Among terpenes, the sesquiterpenes (C15 ) are the largest subclass with bioactivities ranging from aroma to health promotion. In this article, we show a gram-scale production of the sesquiterpene α-humulene in final aqueous concentrations of 2 g L-1 with the recombinant strain Cupriavidus necator pKR-hum in a fed-batch mode on fructose as carbon source and n-dodecane as an extracting organic phase for in situ product removal. Since C. necator is capable of both heterotrophic and autotrophic growth, we additionally modeled the theoretically possible yields of a heterotrophic versus an autotrophic process on CO2 in industrially relevant quantities. We compared the cost-effectiveness of both processes based on a production of 10 t α-humulene per year, with both processes performing equally with similar costs and gains. Furthermore, the expression and activity of 3-hydroxymethylglutaryl-CoA reductase (hmgR) from Myxococcus xanthus was identified as the main limitation of our constructed C. necator pKR-hum strain. Thus, we outlined possible solutions for further improvement of our production strain, for example, the replacement of the hmgR from M. xanthus by a plant-based variant to increase α-humulene production titers in the future.

Cell clarification represents a major challenge for the intensification through very high cell density in the production of biopharmaceuticals such as monoclonal antibodies (mAbs). The present report proposes a solution to this challenge in a streamlined process where cell clarification and mAb capture are performed in a single step using magnetic beads coupled with protein A. Capture of mAb from non-clarified CHO cell suspension showed promising results; however, it has not been demonstrated that it can handle the challenge of very high cell density as observed in intensified fed-batch cultures. The performances of magnetic bead-based mAb capture on non-clarified cell suspension from intensified fed-batch culture were studied. Capture from a culture at density larger than 100 × 106 cells/ml provided an adsorption efficiency of 99% and an overall yield of 93% with a logarithmic host cell protein (HCP) clearance of ≈2–3 and a resulting HCP concentration ≤≈5 ppm. These results show that direct capture from very high cell density cell suspension is possible without prior processing. This technology, which brings significant benefits in terms of operational cost reduction and performance improvements such as low HCP, can be a powerful tool alleviating the challenge of process intensification. QC 20221102

Baicalein and scutellarein are bioactive flavonoids isolated from the traditional Chinese medicine Scutellaria baicalensis Georgi; however, there is a lack of effective strategies for producing baicalein and scutellarein. In this study, we developed a sequential self-assembly enzyme reactor involving two enzymes in the baicalein pathway with a pair of protein-peptide interactions in E. coli. These domains enabled us to optimize the stoichiometry of two baicalein biosynthetic enzymes recruited to be an enzymes complex. This strategy reduces the accumulation of intermediates and removes the pathway bottleneck. With this strategy, we successfully promoted the titer of baicalein by 6.6-fold (from 21.6 to 143.5 mg/L) and that of scutellarein by 1.4-fold (from 84.3 to 120.4 mg/L) in a flask fermentation, respectively. Furthermore, we first achieved the de novo biosynthesis of baicalein directly from glucose, and the strain was capable of producing 214.1 mg/L baicalein by fed-batch fermentation. This work provides novel insights for future optimization and large-scale fermentation of baicalein and scutellarein.

The reliable and cost-efficient manufacturing of monoclonal antibodies (mAbs) is essential to fulfil their ever-growing demand. Cell death in bioreactors reduces productivity and product quality, and is largely attributed to apoptosis. In perfusion bioreactors, this leads to the necessity of a bleed stream, which negatively affects the overall process economy. To combat this limitation, death-resistant Chinese hamster ovary cell lines were developed by simultaneously knocking out the apoptosis effector proteins Bak1, Bax, and Bok with CRISPR technology. These cell lines were cultured in fed-batch and perfusion bioreactors and compared to an unmodified control cell line. In fed-batch, the death-resistant cell lines showed higher cell densities and longer culture durations, lasting nearly a month under standard culture conditions. In perfusion, the death-resistant cell lines showed slower drops in viability and displayed an arrest in cell division after which cell size increased instead. Pertinently, the death-resistant cell lines demonstrated the ability to be cultured for several weeks without bleed, and achieved similar volumetric productivities at lower cell densities than that of the control cell line. Perfusion culture reduced fragmentation of the mAb produced, and the death-resistant cell lines showed increased glycosylation in the light chain in both bioreactor modes. These data demonstrate that rationally engineered death-resistant cell lines are ideal for mAb production in perfusion culture, negating the need to bleed the bioreactor whilst maintaining product quantity and quality.

Refining the energetic costs of cellular maintenance is essential for predicting microbial growth and survival in the environment. Here, we evaluate a simple batch culture method to quantify energy partitioning between growth and maintenance using microcalorimetry and thermodynamic modeling. The constants derived from the batch culture system were comparable to those that have been reported from meta-analyses of data derived from chemostat studies. The model accurately predicted temperature-dependent biomass yield and the upper temperature limit of growth for Desulfovibrio alaskensis G20, suggesting the method may have broad application. An Arrhenius temperature dependence for the specific energy consumption rate, inferred from substrate consumption and heat evolution, was observed over the entire viable temperature range. By combining this relationship for specific energy consumption rates and observed specific growth rates, the model describes an increase in nongrowth associated maintenance at higher temperatures and the corresponding decrease in energy available for growth. This analytical and thermodynamic formulation suggests that simply monitoring heat evolution in batch culture could be a useful complement to the recognized limitations of estimating maintenance using extrapolation to zero growth in chemostats.

Fed-batch culture of Chinese hamster ovary (CHO) cells remains the most commonly used method for producing biopharmaceuticals. Static CHO cell-line engineering approaches have incrementally improved productivity, growth and product quality through permanent knockout of genes with a negative impact on production, or constitutive overexpression of genes with a positive impact. However, during fed-batch culture, conditions (such as nutrient availability) are continually changing. Therefore, traits that are most beneficial during early-phase culture (such as high growth rate) may be less desirable in late phase. Unlike with static approaches, dynamic cell line engineering strategies can optimise such traits by implementing synthetic sense-and-respond programmes. Here, we review emerging synthetic biology tools that can be used to build dynamic, self-regulating CHO cells, capable of detecting intra-/extracellular cues and generating user-defined responses tailored to the stage-specific needs of the production process.

Mathematical modeling is a powerful and inexpensive approach to provide a quantitative basis for improvements that minimize the negative effects of bioreactor heterogeneity. For a model to accurately represent a heterogeneous system, a flow model that describes how mass is channeled between different zones of the bioreactor volume is necessary. In this study, a previously developed compartment model approach based on data from flow-following sensor devices was further developed to account for dynamic changes in volume and flow rates and thus enabling simulation of the widely used fed-batch process. The application of the dynamic compartment model was demonstrated in a study of an industrial fermentation process in a 600 m3 bubble column bioreactor. The flow model was used to evaluate the mixing performance by means of tracer simulations and was coupled with reaction kinetics to simulate concentration gradients in the process. The simulations showed that despite the presence of long mixing times and significant substrate gradients early in the process, improving the heterogeneity did not lead to overall improvements in the process. Improvements could, however, be achieved by modifying the dextrose feeding profile.

Microbial experimental evolution allows studying evolutionary dynamics in action and testing theory predictions in the lab. Experimental evolution in chemostats (i.e. continuous flow through cultures) has recently gained increased interest as it allows tighter control of selective pressures compared to static batch cultures, with a growing number of efforts to develop systems that are easier and cheaper to construct. This protocol describes the design and construction of a multiplexed chemostat array (dubbed “mesostats”) designed for cultivation of algae in 16 concurrent populations, specifically intended for studying adaptation to herbicides. We also present control data from several experiments run on the system to show replicability, data illustrating the effects of common issues like leaks, contamination and clumps, and outline possible modifications and adaptations of the system for future research.

Ansamitocin (AP-3) is an ansamycins antibiotic isolated from Actinosynnema pretiosum and demonstrating high anti-tumor activity. To improve AP-3 production, the A. pretiosum ATCC 31565 strain was treated with atmospheric and room temperature plasma (ARTP). Four stable mutants were obtained by ARTP, of which the A. pretiosum L-40 mutant produced 242.9 mg/L AP-3, representing a 22.5% increase compared to the original wild type strain. With seed medium optimization, AP-3 production of mutant L-40 reached 307.8 mg/L; qRT-PCR analysis revealed that AP-3 biosynthesis-related gene expression was significantly up-regulated under optimized conditions. To further improve the AP-3 production, genome shuffling (GS) technology was used on the four A. pretiosum mutants by ARTP. After three rounds of GS combined with high-throughput screening, the genetically stable recombinant strain G3-96 was obtained. The production of AP-3 in the G3-96 strain was 410.1 mg/L in shake flask cultures, which was 44.5% higher than the L-40 production from the parental strain, and AP-3 was increased by 93.8% compared to the wild-type A. pretiosum. These results suggest that the combination of mutagenesis, seed medium optimization, and GS technology can effectively improve the AP-3 production capacity of A. pretiosum and provide an enabling methodology for AP-3 industrial production.

Bioconversion of glycerol to 1,3-propanediol is a promising way to mitigate the shortage of energy. To maximize the production of 1,3-propanediol, it needs to control precisely microbial fermentation process. However, it might consume lots of human and material resources when conducting experimental tests many times. In this study, a nonlinear enzyme-catalytic dynamical system is developed to describe the bioconversion process of glycerol to 1,3-propanediol, especially continuous piecewise linear functions are used as identification parameters. The existence, uniqueness and continuity of solutions are also discussed. Then, considering the fact that the concentration of intracellular substances is difficult to measure in experiments, a new quantitative definition of biological robustness is introduced as a performance index to determine the identification parameters related to intracellular substances. Meanwhile, a two-phase optimization algorithm is constructed to solve the identification model. By comparison with the experimental data, it can be found that the present nonlinear dynamical system can describe the fermentation process very well. Finally, the present nonlinear dynamical system and the corresponding optimal identification parameters might be useful in future studies on the batch culture of glycerol to 1,3-propanediol.

Nowadays, artificial construction of bacteria-algae consortia to enhance microalgal biomass is prevalent in enclosed systems, while few are built in an open culture. In this study, Achromobacter sp. and Rhizobium sp., isolated from an open pond of Chlorella sorokiniana, were the microalgal growth-promotion bacteria and selected to build the bacteria-algae consortia with axenic C. sorokiniana in open cultivation systems. To examine the performance of these two artificial bacteria-algae consortia in open culture under stable cultivation conditions, the co-cultivation experiments were conducted under constant temperature and light intensity indoors. It was found that Achromobacter sp. gradually lost the dominance of the population in the co-culture and failed to promote the growth of C. sorokiniana during open cultivation. However, the Rhizobium sp. maintained its dominant population of bacterial community in open culture and could promote the growth of C. sorokiniana, with an enhancement of 13.76%. To further evaluate the effects of Rhizobium sp. on microalgae under variations of temperature and sunlight intensity conditions, the open co-cultivation experiments were built outdoors. Results showed that the growth of C. sorokiniana could rise 13.29% only when Rhizobium sp. was added to the culture continuously, and addition of bacterial solution in log-phase of microalgae could help Rhizobium sp. dominate in the bacterial community. In this way, addition of Rhizobium sp. in the log-phase of C. sorokiniana should be an effective process to be applied to open ponds cultivation. Our findings are a step toward applying growth-promotion bacteria for C. sorokiniana for applications in open cultivation systems.

The cell culture is the central piece of a biotechnological industrial process. It includes upstream (e.g. media preparation, fixed costs, etc.) and downstream steps (e.g. product purification, waste disposal, etc.). In the continuous mode of cell culture, a constant flow of fresh media replaces culture fluid until the system reaches a steady state. This steady state is the standard operation mode which, under very general conditions, is a function of the ratio between the cell density and the dilution rate and depends on the media supplied to the culture. To optimize the production process it is widely accepted that the concentration of the metabolites in this media should be carefully tuned. A poor media may not provide enough nutrients to the culture, while a media too rich in nutrients may be a waste of resources because, either the cells do not use all of the available nutrients, or worse, they over-consume them producing toxic byproducts. In this work, we show how an in-silico study of a genome scale metabolic network coupled to the dynamics of a chemostat could guide the strategy to optimize the media to be used in a continuous process. Given a known media we model the concentrations of the cells in a chemostat as a function of the dilution rate. Then, we cast the problem of optimizing the production process within a linear programming framework in which the goal is to minimize the cost of the media keeping fixed the cell concentration for a given dilution rate in the chemostat. We evaluate our results in two metabolic models: first a simplified model of mammalian cell metabolism, and then in a realistic genome-scale metabolic network of mammalian cells, the Chinese Hamster Ovary (CHO) cell line. We explore the latter in more detail given specific meaning to the predictions of the concentrations of several metabolites. This article is protected by copyright. All rights reserved.

Maleate is one of the most important unsaturated four-carbon dicarboxylic acids. It serves as an attractive building block in cosmetic, polymer, and pharmaceutical industries. Currently, industrial production of maleate relies mainly on chemical synthesis using benzene or butane as the starting materials under high temperature, which suffers from strict reaction conditions and low product yield. Here, we propose a novel biosynthetic pathway for maleate production in engineered Escherichia coli. We screened a superior salicylate 5-hydroxylase that can catalyze hydroxylation of salicylate into gentisate with high conversion rate. Then, introduction of salicylate biosynthetic pathway and gentisate ring cleavage pathway allowed the synthesis of maleate from glycerol. Further optimizations including enhancement of precursors supply, disruption of competing pathways, and construction of a pyruvate recycling system, boosted maleate titer to 2.4 ± 0.1 g/L in shake flask experiments. Subsequent scale-up biosynthesis of maleate in a 3-L bioreactor under fed-batch culture conditions enabled the production of 14.5 g/L of maleate, indicating a 268-fold improvement compared with the titer generated by the wildtype E. coli strain carrying the entire maleate biosynthetic pathway. This study provided a promising microbial platform for industrial level synthesis of maleate, and demonstrated the highest titer of maleate production in microorganisms so far.

For the stress from fermenters, downstream processing equipment, and wastewater treatment to be alleviated, lowering salt-dependence in the ectoine synthesis process is of great significance in the moderately halotolerant Halomonas hydrothermalis Y2. In H. hydrothermalis Y2, the σ70- and σ38-controlled promoters of ectA are predicted to be involved in the osmotic regulation of ectoine synthesis. By substituting the ectA promoter with a promoter P265 that identified in the outer membrane pore protein E of H. hydrothermalis Y2, the salt dependence of ectoine synthesis was significantly decreased. In the 500-ml flask containing various NaCl contents, the engineered strain (p/Y2/△ectD/△doeA) showed a remarkably enhanced ability in ectoine synthesis, especially under lower saline stress. After a 36-h fed-batch fermentation in the 1-l fermenter, p/Y2/△ectD/△doeA synthesized 11.5 g ectoine l−1 in the presence of 60 g NaCl−1 l, with a high 0.32 g ectoine l−1 h−1 productivity, a specific productivity of 512.2 mg ectoine per g cell dry weight (CDW)−1, and an excretion ratio of 67 % ectoine. As no impaired growth was observed in strain p/Y2/△ectD/△doeA while ectoine synthesis was increased, this promoter engineering strategy provides a practical protocol for lowering the salt-dependence of ectoine synthesis in this moderately halotolerant strain.

Polyhydroxyalkanoates (PHAs) have attracted attention as an environmentally degradable bioplastic which potentially replaces synthetic polymers used in a wide range of industries. One of most promising microorganisms for the production of PHAs is Pseudomonas putida. In this study, we purpose to develop sustainable processes to convert abundant palm oil available in local market to high value PHAs and optimize PHAs production by Pseudomonas putida TISTR 1522 from saponified palm oil. We found that the highest yield of PHAs production (0.95 g/L, 40.15%) was obtained in culture medium supplemented with 1% (w/v) fatty acid salt by P. putida TISTR 1522 after 24-h cultivation. The intracellular PHAs were located in granules inside the cells, which fluoresced bright yellow by staining with Nile red. The physical appearance of intracellular PHAs investigated by transmission electron microscope (TEM) revealed that PHAs accumulate in granules, about 3–10 granules per cell. These granules are white and roundish-shaped with 0.3–0.5-μm diameter. The 1H NMR spectrum represented the typical characters of medium-chain length-PHAs. This variation of all parameters was successfully demonstrated a good intracellular PHAs accumulation in P. putida TISTR 1522 by fatty acid salt utilization.

Most of the current industrial processes for L-leucine production are based on fermentation, usually in fed-batch operation mode. Although the culture technology has advanced in recent decades, the process still has significant drawbacks. To solve these problems, we investigated the effects of chemostat culture conditions on the production of L-leucine by Corynebacterium glutamicum CP. The dilution rate, the nitrogen source, and the carbon–nitrogen ratio of the medium were optimized. With the addition of ammonium acetate to the chemostat medium, the initial C/N ratio was adjusted to 57.6, and the L-leucine titer reached the highest level at the optimal dilution rate of 0.04 h−1. Compared with fed-batch culture, the L-leucine titer was reduced (from 53.0 to 24.8 g L−1), but the yield from glucose was increased by 10.0% (from 0.30 to 0.33 mol mol−1) and productivity was increased by 58.3% (from 1.2 to 1.9 g L−1 h−1). Moreover, the titer of the by-product L-alanine was significantly reduced (from 8.9 to 0.8 g L−1). In addition, gene expression levels and activity of key enzymes in the synthesis of L-leucine and L-alanine were analyzed to explain the difference of production performance between chemostat culture and fed-batch culture. The results indicate that chemostat culture has great potential to increase the industrial production of L-leucine compared to current fed-batch approaches., Graphical abstract

Violacein is a naturally occurring anticancer therapeutic compound with deep purple color. In this work, we harnessed the modular and combinatorial feature of a Golden Gate assembly method to construct a library of violacein producing strains in the oleaginous yeast Yarrowia lipolytica, where each gene in the violacein pathway was controlled by three different promoters with varying transcriptional strength. After optimizing the linker sequence and the Golden Gate reaction, we achieved high transformation efficiency and obtained a panel of representative Y. lipolytica recombinant strains. By evaluating the gene expression profile of 21 yeast strains, we obtained three colorful compounds in the violacein pathway: green (proviolacein), purple (violacein), and pink (deoxyviolacein). Our results indicated that strong expression of VioB, VioC, and VioD favors violacein production with minimal byproduct deoxyvioalcein in Y. lipolytica, and high deoxyviolacein production was found strongly associated with the weak expression of VioD. By further optimizing the carbon to nitrogen ratio and cultivation pH, the maximum violacein reached 70.04 mg/L with 5.28 mg/L of deoxyviolacein in shake flasks. Taken together, the development of Golden Gate cloning protocols to build combinatorial pathway libraries, and the optimization of culture conditions set a new stage for accessing the violacein pathway intermediates and engineering violacein production in Y. lipolytica. This work further expands the toolbox to engineering Y. lipolytica as an industrially relevant host for plant or marine natural product biosynthesis.

Bioprocess modeling has become a useful tool for prediction of the process future with the aim to deduce operating decisions (e.g. transfer or feeds). Due to variabilities, which often occur between and within batches, updating (re-estimation) of model parameters is required at certain time intervals (dynamic parameter estimation) to obtain reliable predictions. This can be challenging in the presence of low sampling frequencies (e.g. every 24 h), different consecutive scales and large measurement errors, as in the case of cell culture seed trains. This contribution presents an iterative learning workflow which generates and incorporates knowledge concerning cell growth during the process by using a moving horizon estimation (MHE) approach for updating of model parameters. This estimation technique is compared to a classical weighted least squares estimation (WLSE) approach in the context of model updating over three consecutive cultivation scales (40–2160 L) of an industrial cell culture seed train. Both techniques were investigated regarding robustness concerning the aforementioned challenges and the required amount of experimental data (estimation horizon). It is shown how the proposed MHE can deal with the aforementioned difficulties by the integration of prior knowledge, even if only data at two sampling points are available, outperforming the classical WLSE approach. This workflow allows to adequately integrate current process behavior into the model and can therefore be a suitable component of a digital twin. Bioprocess modeling has become a useful tool for prediction of the process future with the aim to deduce operating decisions (e.g. transfer or feeds). Due to variabilities, which often occur between and within batches, updating (re-estimation) of model parameters is required at certain time intervals (dynamic parameter estimation) to obtain reliable predictions. This can be challenging in the presence of low sampling frequencies (e.g. every 24 h), different consecutive scales and large measurement errors, as in the case of cell culture seed trains. This contribution presents an iterative learning workflow which generates and incorporates knowledge concerning cell growth during the process by using a moving horizon estimation (MHE) approach for updating of model parameters. This estimation technique is compared to a classical weighted least squares estimation (WLSE) approach in the context of model updating over three consecutive cultivation scales (40–2160 L) of an industrial cell culture seed train. Both techniques were investigated regarding robustness concerning the aforementioned challenges and the required amount of experimental data (estimation horizon). It is shown how the proposed MHE can deal with the aforementioned difficulties by the integration of prior knowledge, even if only data at two sampling points are available, outperforming the classical WLSE approach. This workflow allows to adequately integrate current process behavior into the model and can therefore be a suitable component of a digital twin.

In order to increase content of glucuronic acid in the exopolysaccharide (EPS) and its flocculating activity, an UDP-glucose dehydrogenase gene was overexpressed in Lipomyces starkeyi V19. The obtained U9 strain could produce 62.1 ± 1.2 g/l EPS while the V19 strain only produced 53.5 ± 1.3 g/l EPS. The compositions of monosaccharides (mannose, glucuronic acid and galactose) in the purified EPS (U9-EPS) from the U9 strain contained 3.79:1:5.52 while those in the purified EPS (V19-EPS) were 3.94:1:6.29. The flocculation rate of the U9-EPS on kaolin clay reached 87.9%, which was significantly higher than that (74.7%) of the V19-EPS while the decolorization rate of Congo Red (CR) by the U9-EPS reached 94.3%, which was significantly higher than that of CR by the V19-EPS (86.23%). The results showed that the purified bioflocculant U9-EPS had effective flocculation of kaolin clay. The U9-EPS also had high ability to flocculate the polluted river water and decolorize Congo red.