Shake Cultures Research Articles

Improved oxygenation dramatically alters metabolism and gene expression in cultured primary mouse hepatocytes.

Primary hepatocyte culture is an important in vitro system for the study of liver functions. In vivo, hepatocytes have high oxidative metabolism. However, oxygen supply by means of diffusion in in vitro static cultures is much less than that by blood circulation in vivo. Therefore, we investigated whether hypoxia contributes to dedifferentiation and deregulated metabolism in cultured hepatocytes. To this end, murine hepatocytes were cultured under static or shaken (60 revolutions per minute) conditions in a collagen sandwich. The effect of hypoxia on hepatocyte cultures was examined by metabolites in media and cells, hypoxia‐inducible factors (HIF)‐1/2α western blotting, and real‐time quantitative polymerase chain reaction for HIF target genes and key genes of glucose and lipid metabolism. Hepatocytes in shaken cultures showed lower glycolytic activity and triglyceride accumulation than static cultures, compatible with improved oxygen delivery and mitochondrial energy metabolism. Consistently, static cultures displayed significant HIF‐2α expression, which was undetectable in freshly isolated hepatocytes and shaken cultures. Transcript levels of HIF target genes (glyceraldehyde 3‐phosphate dehydrogenase [Gapdh], glucose transporter 1 [Glut1], pyruvate dehydrogenase kinase 1 [Pdk1], and lactate dehydrogenase A [Ldha]) and key genes of lipid metabolism, such as carnitine palmitoyltransferase 1 (Cpt1), apolipoprotein B (Apob), and acetyl‐coenzyme A carboxylase 1 (Acc1), were significantly lower in shaken compared to static cultures. Moreover, expression of hepatocyte nuclear factor 4α (Hnf4α) and farnesoid X receptor (Fxr) were better preserved in shaken cultures as a result of improved oxygen delivery. We further revealed that HIF‐2 signaling was involved in hypoxia‐induced down‐regulation of Fxr. Conclusion: Primary murine hepatocytes in static culture suffer from hypoxia. Improving oxygenation by simple shaking prevents major changes in expression of metabolic enzymes and aberrant triglyceride accumulation; in addition, it better maintains the differentiation state of the cells. The shaken culture is, therefore, an advisable strategy for the use of primary hepatocytes as an in vitro model. (Hepatology Communications 2018;2:299‐312)

Enhanced expression of a biosimilar monoclonal antibody with a novel NS0 platform.

The precise product quality and lower cost of goods demands of the growing biosimilars industry are driving biomanufacturing innovation. Biosimilar cell lines that produce complex glycoproteins such as monoclonal antibodies must be both highly productive and express a product with critical quality attributes closely matching those of the innovator reference. In this work, a biomanufacturing platform is described that harnesses the commercially-established NS0 host cell in new ways to create stable, highly productive cell lines with characteristics meeting the current demands. A cholesterol metabolic selection marker and implementation strategy that can be generically applied are shown to yield high expressing cell lines as well as eliminate the need for cholesterol addition, which has been a significant barrier in both stainless steel reactors as well as in single-use plastic systems. Additionally, for the first time, a multiplex selection strategy was implemented that served to increase NS0 cell line specific productivity >10-fold and volumetric yields >6-fold. The best overall performing cell line had a Qp of 28.5 picograms per cell per day and was rapidly adapted to a lean production medium. Yields in l-glutamine fed-batch shaker cultures exceeded 500mg/L. An initial screening of four feeding strategies resulted in a final 13-day yield of over 1.4g/L in small shaker culture. Overall, this work shows both the strategy to develop biosimilar cell lines and the commercial potential of a novel expression system highly suited for the manufacture of biosimilars of reference biologics currently produced in murine cells. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:455-462, 2018.

Characteristics of Staphylococcal Enterotoxin A Production and Growth of Staphylococcus aureus in Shaking and Stationary Cultures.

Staphylococcal food poisoning, which is still a serious health problem worldwide, is caused by staphylococcal enterotoxin (SE) . Among many types of SE, staphylococcal enterotoxin type A (SEA) is known to be the most responsible for staphylococcal food poisoning. Production by SEA-producing strains in shaking culture has been studied by many investigators, whereas the kinetical differences between shaking and stationary cultures have not been studied intensively. Therefore, this difference was studied at various temperatures from 14℃ to 46℃ in the present study. Consequently the maximum SEA concentration and populations of SEA producer in shaking culture was higher than those in stationary culture. Of interest, however, the productivity, which is the maximum SEA amount produced by one cell, in shaking cultures was lower than that in stationary culture. Kinetic analysis clarified that SEA gene expression in staphylococcal cells preceded toxin production at optimal temperature. Next, several SEA producers were studied for the maximum toxin production at various temperatures from 14℃ to 42℃ in shaking culture. Consequently all strains showed different patterns, suggesting that the characteristics of SEA production of these strains would be strain-specific. These results in this study would provide useful, basic information to prevent staphylococcal food poisoning outbreaks.

Increase of Citric Acid Production by Aspergillus niger InaCC F539 in Sorghum's Juice Medium Amended with Methanol

ABSTRACT Citric acid demand increases steadily, and there is a need to increase productivity through selection of suitable carbon sources, and addition of substances that increase citric acids production rate. Methanol has been suggested to increase citric acid fermentation on high carbohydrate containing substances. The objective of the study was to evaluate the suitability of sweet sorghum juice for citric acids production and to verify the effect of methanol on citric acids production using Aspergillus niger InaCC F539 as inoculant. Sweet sorghum juice with the total initial reducing sugar of 11.5 % (w/v) was used as the sole carbon sources. To study the effect of total initial reducing sugar on citric acid production the initial reducing sugar was adjusted to the concentration of 30 to 75 g/L. Preliminary experiment was conducted to get the optimum methanol concentration that stimulate citric acid production. The optimum methanol concentration that stimulate citric acid production was 4% (v/v). Submerged fermentation was conducted as shake culture (125 rpm at 28 °C). Citric acids production was affected by total initial reducing sugar. Higher total initial reducing sugar produced higher citric acids. Maximum citric acid production was 18.96g/L on sweet sorghum juice with 75 g/L total initial reducing sugar. Methanol 4 % (v/v) increase citric acid production by 41.35 to 65.89 %. Juice of sweet sorghum was a good medium for citric acids production, and methanol stimulate and increase citric acid production. It is a good basis for exploring efficient and cost effective industrial scale citric acid production. Keywords: Citric acid, Methanol, Sweet sorghum, Aspergillus niger

The role of poloxamer 188 for cord blood mononuclear cells into megakaryocytes cultivation and induction in three-dimensional WAVE Bioreactor

目的观察泊洛沙姆188（P188）对体外三维（3D）培养诱导脐血单个核细胞向巨核细胞分化的影响。方法将分离的脐血单个核细胞分别接种于细胞瓶和细胞培养袋中，后者采用WIGGENS摇床模拟生物反应器进行3D培养。在巨核细胞诱导培养基中加入P188体外培养14 d，观察细胞形态、计数细胞数并计算细胞存活率，采用流式细胞术观察巨核细胞表面标志表达情况。结果与采用传统的细胞培养瓶二维（2D）培养诱导巨核细胞相比，2D+P188培养组巨核系CD41+、CD41+/CD61+、CD61+细胞数明显增加（P值均<0.01）；在3D培养中加入P188，细胞体积变大，核形状不规则，胞质含紫红色颗粒，细胞分化更接近成熟。2D培养、3D培养及3D+P188培养组组间巨核细胞表面标志CD41、CD41/CD61、CD61表达水平差异有统计学意义（P值均<0.01）。LSD-t检验两两比较显示，与2D培养相比，3D培养诱导巨核细胞存活率及细胞数均降低（P值分别为0.018、0.027），3D+P188培养组细胞数、细胞存活率与2D和3D培养组比较差异均无统计学意义（P值均>0.05）。而3D培养组巨核细胞CD41/CD61表达水平为（36.30±1.27）%，高于2D培养组的（23.95±1.34）%（P=0.002），3D+P188培养组CD41/CD61表达水平更高[（59.45±1.20）%]。结论3D培养有利于巨核系祖细胞诱导分化，但细胞存活率低，加入P188，细胞生存状态好，且诱导效率更高。

Protein Production Using the Baculovirus Expression System.

Baculovirus expression systems are well established as an easy and reliable way to produce high quality recombinant proteins. Baculoviruses can also be used to transduce mammalian cells, termed 'BacMam', with considerable potential in biomedical applications. This chapter explains the process of making a recombinant baculovirus, encompassing production of a recombinant virus by homologous recombination in insect cells, followed by amplification and titration of the virus-all steps needed before commencing gene expression and protein production. We also cover the use of small-scale test expression to provide an initial indication of quality and protein yield. Whereas proteins expressed at high levels can be directly scaled up, more challenging proteins may require optimization of cell lines, growth conditions, or harvest times. Scale-up and purification approaches are discussed, focusing on working with large shake cultures and use of the Wave bioreactor. © 2018 by John Wiley & Sons, Inc.

Improvement of Soil Properties, Growth of Cucumber and Protection against Fusarium Wilt by Piriformospora indica and Two Industrial Organic Wastes

The current work was focused on characterizing bagasse ash (BA) and press mud (PM) as soil amendments and to study their effect in combination with the endophytic fungus Piriformospora indica on Fusarium wilt (FW) of cucumber caused by Fusarium oxysporum f. sp. cucumerinum (Fo). Whereas BA and PM improved almost all physico-chemical properties of the soil evaluated, seed treatment with P. indica had no such effect. In shake culture in potato dextrose broth (PDB) medium amended with aqueous extracts of BA and PM, alone or in combination, production mycelial mass of Fo was significantly decreased by PM extract, while production mycelial mass of P. indica was highly improved. The colonization rate of cucumber roots by P. indica as determined by microscopy was highly increased by increasing amounts of BA, PM and BA+PM added to the soil. Seed treatment of cucumber with P. indica before plant cultivation in non-amended soil significantly decreased the disease severity of FW and improved plant growth. When seed treated with P. indica was sown into soil amended with BA, PM or the combination of both, the disease severity was even more reduced than after seed treatment with P. indica alone. In this respect, amendment with PM was more effective than with BA, and the combinations were more effective than the single treatments. Hence, there is a scope to integrate PM and BA as soil amendments in combination with P. indica for eco-friendly FW management, improving soil properties and growth of cucumber plants.

Recombinant expression insulin-like growth factor 1 in Bacillus subtilis using a low-cost heat-purification technology

Abstract Insulin-like growth factor 1 (IGF-1) has many positive effects on neonates’ intestinal health. However, the production of IGF-1 involves complex and costly chromatography-based purification devices that greatly increase the cost of IGF-1. In this study, we applied the DAMP4 fusion technology to express IGF-1 in Bacillus subtilis cell factories without using these purification devices. After optimizing the induction conditions in a shaker culture procedure, Western blotting results indicated that the DAMP4-IGF-1 fusion protein was successfully secreted into the culture medium. Approximately 17 mg/L DAMP4-IGF-1 was obtained by a simple heat-purification method. The subsequent cleavage of purified fusion protein by enterokinase produced approximately 2.5 mg/L of tag-free recombinant IGF-1 with immunogenic activity. Additionally, the recombinant IGF-1 had the biological activity to promote the proliferation of Caco-2 cells. This approach provides a safe, versatile and cost-effective protocol to produce milkborne growth factors in B. subtilis as well as other functional low molecular weight peptide products.

KAJIAN RANCANGAN UNDANG-UNDANG PEMBERANTASAN TINDAK PIDANA TERORISME

At this time, Indonesian revising for the anti terrorism number 15 year 2003. It’s cause pro and contra about the draft of articles. The issue that often turn up is the anti terrorism article drafts should more against terrorism crime and all at once human right respectful.This article study about interpretation of article drafts and comparison with the anti terrorism number 15 year 2003. So, the more and less of article drafts will be knowing. Revise for the anti terrorism number 15 year 2003 hoping can find strategic and tactis to overcome terrorism crime without shake Indonesian social and culture system.

A method to increase the production of insulin precursor in Pichia pastoris

To improve the yield of insulin precursor (PI), we constructed a recombinant expression vector pPIC9K-PI and transformed it into Pichia pastoris GS115 using electroporation. After screening, a mutant strain CL012 with 12 copies was obtained on the YPDS plate containing 4.0 mg/mL G418. Then, the components of SNAREs (Soluble N-ethylmaleimide-sensitive factor attachment receptor proteins), SNC2 and SNC2-SSO2, were expressed in the strain CL012 to explore the effect of SNAREs on the yield of PI. In shake flask culture, the strains expressing SNC2 and SNC2-SSO2 yielded PI of 1.89 mg/L and 2.21 mg/L after methanol induction for 96 h, which were improved by 23.53% and 44.44% compared to that of strain CL012 (1.53 mg/L), respectively. Further, in a 5-L bioreactor, the yield of PI with strain CL012 was 53 mg/L for high-density fermentation, after 96 h of methanol induction, which was 34.64-fold higher than that of shake culture. The strains expressing SNC2 and SNC2-SSO2 yielded the PI of 64 mg/L and 78 mg/L, which were respectively increased by 20.75% and 47.17%, compared to that of strain CL012. This work indicated that SNAREs components promoted the secretion of PI to improve its heterologous expression in P. pastoris.

Development and Characterization of Polymer Eco-Composites Based on Natural Rubber Reinforced with Natural Fibers.

Natural rubber composites filled with short natural fibers (flax and sawdust) were prepared by blending procedure and the elastomer cross-linking was carried out using benzoyl peroxide. The microbial degradation of composites was carried out by incubating with Aspergillus niger recognized for the ability to grow and degrade a broad range of substrates. The extent of biodegradation was evaluated by weight loss and cross-linking degree study of composites after 2 months incubation in pure shake culture conditions. Scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FT-IR) have proved to be precious and valuable instruments for morphological as well as structural characterization of the composites before and after incubation with Aspergillus niger.

Reply to the Comment on "Melanisation of Aspergillus terreus-Is Butyrolactone I Involved in the Regulation of Both DOPA and DHN Types of Pigments in Submerged Culture? Microorganisms 2017, 5, 22".

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Rapid screening and construction of high-efficient petroleum-degrading microflora through enzyme activity assay

It is of great practical significance to rapidly screen the mixed microbial flora for degradation of complex organic pollutants. It was the first time to rapidly calibrate the petroleum hydrocarbon degradation ability of highly efficient degrading microflora by measuring enzyme activity analyzed by the MicroRespTM technology. The results were validated by the traditional shaking culture. According to the activity analysis of exoenzymes, endoenzymes and mixed enzymes, the degradation abilities of microflora and enzymes (including exoenzymes, endoenzymes and mixed enzymes) on petroleum hydrocarbon was investigated. The results showed that the enzyme activity assay had perfect sensitivity and high-throughput for rapidly detecting the degradation ability of the petroleum hydrocarbon. The test period was 12 h which was 10 times shorter than shaking culture for 120 microflora combinations. The maximum degradation extent of the petroleum hydrocarbon by microflora reached (56.1±1.6)%. It was concluded that the method of enzyme activity assay was high-efficient and high-throughput screening, which could be used in the construction of petroleum-degrading microflora.

Liquid shake culture overcomes solid plate culture in inducing conidial production of Colletotrichum isolates

Species of fungi in the genus Colletotrichum cause damaging anthracnose diseases on many economically important fruit, vegetable, and ornamental crops. Unfortunately, the fact that many isolates fail to sporulate on artificial media limits research requiring conidia. We found that liquid culture efficiently rescued conidial production of Colletotrichum isolates being impotent to do so on solid plate media, with the yield averaging 107 per mL after four days. We determined that the most efficient culture protocol was potato dextrose broth (PDB) shake culture (140 rpm) at 25 °C. In addition, it was essential that enough medium was used to cover the mycelial balls. The method was applicable to different Colletotrichum species from a range of hosts. In sum, we report here that liquid shake culture offers an alternative in inducing conidia production of ‘non-sporulating’ Colletotrichum isolates, a critical step for many research activities in the lab.

Accumulation of biomass and four triterpenoids in two-stage cultured Poria cocos mycelia and diuretic activity in rats

Poria cocos (Schw.) Wolf, an important medicinal and food fungus, is well known in East Asia. Due to growing market demand, long cultivation period, and consumption of pine trunk during cultivation, developing alternative methods for producing P. cocos and/or its active components is of interest. In the present study, the effects of different culture methods on biomass and accumulation of four triterpenoids were investigated. The ethanol extract of fermented mycelium (EFM) was orally administered to rats. Urine output and concentrations of electrolytes (Na+, K+, and Cl−) were measured. Our results showed that mycelia grew better under continuous shaking culture condition (7.5 g DW·L−1), and higher triterpenoid levels were accumulated in two-stage culture (112 mg·L−1, 2.03%). The optimal starting time of static culture for triterpenoid yield was 4th d after shaking culture. Single administration of middle and high dose of EFM significantly increased urine output, Na+ and Cl− excretion, and Na+/K+ ratio. These results suggested that ethanol extract of cultured mycelia showed significant diuretic activity in rats and two-stage culture of P. cocos could be an alternative way to produce mycelia and triterpenoids.

Optimization of expression and purification of recombinant S1 domain of the porcine epidemic diarrhea virus spike (PEDV- S1) protein in Escherichia coli

ABSTRACTThe PEDV-S1 protein, one of the members of a large family of PEDV, is significantly upregulated and has been targeted as a biomarker of cellular stress in several studies. Herein, conditions were optimized to increase the yield of recombinant PEDV-S1 protein in its native state, primarily focusing on the optimization of upstream processing parameters that lead to an increase in the specific as well as volumetric yield of the protein. The results showed that the production of PEDV-S1 was increased proportionally with increased incubation temperature up to 37 °C. Induction with 10 lM IPTG was sufficient to induce the expression of PEDV-S1 which was 100 times less than the normally used IPTG concentration. Furthermore, the results indicate that induction during early to late exponential phase produced relatively high levels of PEDV-S1 in soluble form. In addition, 5 h of post-induction incubation was found to be optimal to produce folded PEDV-S1 with higher specific and volumetric yield. Subsequently, highly pure and homogenous PEDV-S1 preparation was obtained using metal affinity and size exclusion chromatography. Taken together, the results showed successful production of electrophoretically pure recombinant PEDV-S1 protein in Escherichia coli (E. coli) in milligram quantities from shake flask liquid culture.

Implications of evolutionary engineering for growth and recombinant protein production in methanol-based growth media in the yeast Pichia pastoris

BackgroundPichia pastoris is a widely used eukaryotic expression host for recombinant protein production. Adaptive laboratory evolution (ALE) has been applied in a wide range of studies in order to improve strains for biotechnological purposes. In this context, the impact of long-term carbon source adaptation in P. pastoris has not been addressed so far. Thus, we performed a pilot experiment in order to analyze the applicability and potential benefits of ALE towards improved growth and recombinant protein production in P. pastoris.ResultsAdaptation towards growth on methanol was performed in replicate cultures in rich and minimal growth medium for 250 generations. Increased growth rates on these growth media were observed at the population and single clone level. Evolved populations showed various degrees of growth advantages and trade-offs in non-evolutionary growth conditions. Genome resequencing revealed a wide variety of potential genetic targets associated with improved growth performance on methanol-based growth media. Alcohol oxidase represented a mutational hotspot since four out of seven evolved P. pastoris clones harbored mutations in this gene, resulting in decreased Aox activity, despite increased growth rates. Selected clones displayed strain-dependent variations for AOX-promoter based recombinant protein expression yield. One particularly interesting clone showed increased product titers ranging from a 2.5-fold increase in shake flask batch culture to a 1.8-fold increase during fed batch cultivation.ConclusionsOur data indicate a complex correlation of carbon source, growth context and recombinant protein production. While similar experiments have already shown their potential in other biotechnological areas where microbes were evolutionary engineered for improved stress resistance and growth, the current dataset encourages the analysis of the potential of ALE for improved protein production in P. pastoris on a broader scale.

Osmotic stress in colony and planktonic cells of Pseudomonas putida mt-2 revealed significant differences in adaptive response mechanisms

Planktonic cells and those grown on surfaces (or as colony biofilm) are known to show significant differences regarding growth behavior, cell physiology, gene expression and stress tolerance. In order to compare stress behavior of different growth forms, shake cultures for planktonic growth and agar plate cultivation for colony growth, were carried out with the well investigated model organism, Pseudomonas putida mt-2. Cells were exposed to sodium chloride to cause osmotic stress as one main environmental stressor bacteria have to cope with when growing in soil. Planktonic cells were more tolerant with a complete inhibition of growth at 0.7 M NaCl, compared to 0.5 M for agar-grown cells. Cell surface hydrophobicity, measured as water contact angles, was significantly higher for agar-grown cells (92°) than for planktonic cells (40°), and increased in the presence of NaCl. Agar-grown cells also showed a significantly higher degree of saturation of membrane fatty acids that increased in the presence of NaCl. These results demonstrate that planktonic and colony grown bacteria show different responses when confronted with osmotic stress suggesting that the tolerance and adaptive mechanisms are dependent on the environmental conditions as well as the initial physiological state.

Leaching of rare earth elements from fluorescent powder using the tea fungus Kombucha

In most modern technologies such as flat screens, highly effective magnets and lasers, as well as luminescence phosphors, Rare Earth Elements (REE) are used. Unfortunately no environmentally friendly recycling process exists so far. In comparison to other elements the interaction of microorganisms with REE has been studied to a less extent. However, as REE are ubiquitously present in nature it can be assumed that microorganisms play an important role in the biogeochemistry of REE. This study investigates the potential of organic acid-producing microbes for extracting REE from industrial waste.In Germany, 175 tons of fluorescent phosphor (FP) are collected per year as a distinct fraction from the recycling of compact fluorescent lamps. Because the FP contains about 10% of REE-oxides bound in the so-called triband dyes it is a readily accessible secondary resource of REE. Using the symbiotic mixed culture Kombucha, consisting of yeasts and acetic acid bacteria, REE were leached at a significant rate. The highest leaching-rates were observed in shake cultures using the entire Kombucha-consortium or its supernatant as leaching agent compared to experiments using the isolates Zygosaccharomyces lentus and Komagataeibacter hansenii as leaching organisms. During the cultivation, the pH decreased as a result of organic acid production (mainly acetic and gluconic acid). Thus, the underlying mechanism of the triband dye solubilisation is probably linked to the carboxyl-functionality or a proton excess. In accordance with the higher solubility of REE-oxides compared to REE-phosphates and –aluminates, the red dye Y2O3:Eu2+ containing relatively expensive REE was shown to be preferentially solubilized.These results show that it is possible to dissolve the REE-compounds of FP with the help of microbial processes. Moreover, they provide the basis for the development of an eco-friendly alternative to the currently applied methods that use strong inorganic acids or toxic chemicals.

Production of Biomass by Schizophyllum commune and Its Antifungal Activity towards Rubberwood-Degrading Fungi

Rubberwood is the most popular timber for furniture manufacturing industry in Malaysia. Major drawback concerned that rubberwood is very prone to attack by fungi and wood borers, and the preservation method using boron compounds exhibited hazardous effect to the workers. Fungal-based biological control agents have gained wide acceptance and Schizophyllum commune secondary metabolite played an important role in term of antifungal agent productivity. The effects of initial pH, incubation temperature and agitation on biomass production by S. commune were investigated under submerged shake culture. In this work, it was found that the synthetic medium with initial solution pH of 6.5 and incubated at 30oC with shaking at 150 rpm provided the highest biomass production. The biomass extract from S. commune was then applied onto the rubberwood block panel to investigate its effectiveness. The results showed that biomass extract at a concentration of 5 μg/μL could inhibit the growth of selected rubberwood-degrading fungi, such as Lentinus sp., L. strigosus and Pycnoporus sanguineus.