Biphasic Cultivation Research Articles

Microbial Polyphenol Production in a Biphasic Process

Microbial synthesis of aromatic compounds is generally limited by inherent product toxicity toward the producing cells. Here, in situ extractive strategies represent an efficient approach to avoid such toxic effects and to increase the overall process performance. We conducted a solvent screening to identify suitable organic solvents to develop a biphasic extractive strategy for microbial plant polyphenol production using Corynebacterium glutamicum. From 10 pre-selected organic solvents, tributyrin (TB) showed the best biocompatibility and was chosen for the biphasic extraction process due to its beneficial effect on partitioning and solubility of the plant polyphenol resveratrol. In bioreactors, biphasic cultivation with TB allowed for a product titer of 7.5 mM (1.71 g L–1) resveratrol with a volumetric productivity of 0.26 mM h–1 and a product yield of 0.92 mol mol–1. This biphasic cultivation procedure can be directly employed for the synthesis of other aromatics with similar properties using C. glutamicum.

Isolation, characterization, culturing, and formulation of a new Beauveria bassiana fungus against Diaphorina citri

The Asian citrus psyllid, Diaphorina citri, is a worldwide destructive citrus pest. It aids in the transmission of a group of phloem-limited bacteria (Candidatus Liberibacter spp.) that leads to Huanglongbing (HLB) disease. In this study, a new Chinese native fungal strain (F-HY006) was isolated from the cadavers of D. citri collected from Ganzhou, Jiangxi in China (114°94′ E, 25°87′ N). The isolate was virulent towards adult female D. citri with corrected mortality of 76% and LT50 of 4.09 d after application of a suspension containing 107 conidia/ml (400 µl/citrus seedling). Morphologic and phylogenetic analyses identified this isolate as Beauveria bassiana. Based on its virulence against D. citri and growth characteristics (2.89 ± 0.28 × 105 conidia/cm2) on the PDA plate, biphasic (liquid–solid) cultivation for conidia production of B. bassiana F-HY006 was optimized using several nutritional and environmental factors. Soybean meal + cottonseed powder (1:1) and continuous fluorescent light resulted in the highest conidial production (5.7 × 109/g dry substrate). Moreover, the oil-based conidial formulation was optimized using sesame oil as an effective carrier, which resulted in a significant enhancement of the conidial shelf life, tolerance to thermal stress, and virulence against D. citri compared to unformulated conidia. Therefore, we have prepared a potential mycopesticide for the biological control of D. citri based on the optimized biphasic culture growth and formulation of an isolated virulent fungus, B. bassiana F-HY006.

Biphasic optimization approach for maximization of lipid production by the microalga Chlorella pyrenoidosa.

The aim of the study was to identify the optimum cultivation conditions for the microalgal growth and lipid production of the oleaginous microalga Chlorella pyrenoidosa Chick (IPPAS C2). Moreover, an appropriate NO3- concentration in the cultivation medium for maximized lipid accumulation was determined. The experimental design involved a biphasic cultivation strategy with an initial biomass accumulating phase under optimized light (400μmol/m2 per s), temperature (25°C), and elevated CO2 concentration in the air mixture (3%), followed by a mid-elevated CO2 concentration (0.5%) for lipid induction. The highest lipid yields of 172.47 ± 18.1 and 179.65 ± 25.4mg/L per day were detected for NO3- concentrations of 100 and 150mg/L. The optimization approach presented here led not only to the maximization of lipid yield but also to the development of a biphasic cultivation strategy easily applicable to the cultivation process without the necessity for algal cell harvesting between the first and second cultivation phases.

Accumulation of cyanobacterial oxadiazine nocuolin A is enhanced by temperature shift during cultivation and is promoted by bacterial co-habitants in the culture

Proper setting of cultivation conditions is essential for production of high-value compounds in microbial biotechnology. The present study characterizes photoautotrophic growth and capacity to accumulate the antiproliferative secondary metabolite Nocuolin A (NoA) in cyanobacterium Nostoc sp. CCAP 1453/38. As the cyanobacterial culture was found to be non-axenic, the bacteria accompanying the culture were characterized, then the growth demands and NoA production in the Nostoc-bacterial consortium were determined, and finally an axenic strain was prepared. For the purposes of growth characterization, the culture was maintained in a quasi-continuous regime under various light intensities, temperatures, and inorganic carbon concentrations in a small-scale laboratory photobioreactor. The maximum biomass growth rate obtained was 0.10h−1 (doubling time Dt=6.93h). Following optimal growth conditions were identified: temperature of 35°C, light intensity 600μmol(photons) m−2 s−1, and 2500ppm CO2 in the sparging gas. As the temperature optima for the biomass production and for NoA accumulation differed, biphasic cultivation for maximal NoA yield was designed, leading to a three times more effective cultivation procedure compared to batch culture maintained at a temperature optimal for NoA production. The increased NoA accumulation at reduced temperature that correlated with enhanced expression of NoA biosynthetic genes after the temperature shift suggested its regulation occurs at the expression level. It has further been shown that NoA production is reduced in axenic culture, which indicates that it is also triggered by presence of bacteria. This study shows an example of how a biphasic cultivation mode with different temperatures can be used in high-value compound production processes. It also brings direct evidence that cyanobacterial strain axenization can lead to a rapid decrease in production of valuable compounds and that non-axenic strains may be considered more suitable for retrieval and initial production of novel pharmaceutical leads.

Comparative evaluation of physico-chemical characteristics of biopolyesters P(3HB) and P(3HB-co-3HV) produced by endophytic Bacillus cereus RCL 02

Bacteria endogenously residing within the plant tissues have attracted significant attention for production of biopolyester, polyhydroxyalkanoates (PHAs). Bacillus cereus RCL 02 (MCC 3436), a leaf endophyte of oleaginous plant Ricinus communis L. accumulates 81% poly(3-hydroxybutyrate) [P(3HB)] of its cell dry biomass when grown in mineral salts (MS) medium. The copolymer production efficiency of B. cereus RCL 02 was evaluated in valeric acid supplemented MS medium under biphasic cultivation condition. The copolymer so produced has been compared with the P(3HB) isolated from RCL 02 in terms of thermal, mechanical and chemical properties. Valeric acid supplementation as co-substrate in the medium has led to the production of copolymer of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) [P(3HB-co-3HV)] with 14.6 mol% 3HV. The identity of the polymers has been confirmed by X-ray diffraction (XRD) analysis, Fourier transform infrared (FTIR) and nuclear magnetic resonance (NMR) spectroscopic studies. Thermogravimetric analysis (TGA) revealed that P(3HB) and P(3HB-co-3HV) films degraded at 278.66°C and 273.49°C, respectively. The P(3HB-co-3HV) showed lower melting temperature (165.03°C) compared to P (3HB) (170.74°C) according to differential scanning calorimetry (DSC). Incorporation of 3HV monomers decreased the tensile strength (21.52 MPa), tensile modulus (0.93 GPa), storage modulus (E′) (0.99 GPa) and increased % elongation at break (12.2%) of the copolyester. However, P(3HB) showed better barrier properties with lower water vapor transmission rate (WVTR) of 0.55 g-mil/100 in2/24 h. These findings emphasized exploration of endophytic bacterial strain (RCL 02) to produce biodegradable polyesters which might have significant potential for industrial application.

Genomic analysis of Blastocystis hominis isolates in patients with HIV-positive using locus SSU-rDNA.

Blastocystis hominis (B. hominis) is a protozoan zoonosis which clinical signs of infection with this parasite has been reported to be more severe in patients with weakened immune systems than healthy controls. So, the aim of the study was to evaluate genomic analysis of B. hominis isolates obtained from patients with HIV-positive using locus SSU-rDNA. At first, 268 stool samples were randomly collected from patients with HIV-positive referred to health centers of Khuzestan province, southwest of Iran. Formol-ether and direct smear techniques were used for the detection of parasitic agents. After extracting DNA, the samples were analyzed by the PCR method. Finally, the subtypes were determined by the sequencing and PCR methods. New samples were used for the preparation of positive control sample; they were cultured in coagulant-serum biphasic cultivation media. Of 268 stool samples, 33 (12.3%) cases were detected positive for B. hominis using Formol-Ether technique but 51 (19%) cases were positive using molecular method. The most common isolates were related to the subtype III with 29 positive cases (56.8%), then, genotype I with 11 (21.6%) cases, 6 cases (11.8%) with genotype II, 3 (5.9%) combined cases with genotypes I and III as well as 2 cases (3.9%) with genotype VI. There was a significant difference between two groups of HIV-positive patients (infected with the parasite and/or without the parasite) in the term of the mean of TCD4-positive cells. The results indicated a relatively high prevalence of B. hominis in HIV-positive patients as well as our findings may represent that the number reduction of TCD4-positive cells has an effective role in the increased risk of the parasitic infection in HIV-positive patients.

CHO 세포의 2단계 배양을 통한 Albumin-erythropoietin의 시알산 증대

In glycoprotein, Terminal sialic acid residues of N-linked glycan are imperative things because they prevent the recognition from asialoglycoprotein-receptor that affect the half-life of glycoproteins. So establishment of culture process for enhancing sialic acid is important to maximize sialic acid contents of glycoprotein. In this study, we investigated effects of biphasic culture of Chinese hamster ovary (CHO) cells producing albumin-erythropoietin to increase sialylation. Biphasic cultures were performed with shift of <TEX>$CO_2$</TEX> concentrations and temperatures at day 5 when viable cell density was decreased and sialidase was started to be released by cell lysis. The examined temperature set points were 33, 35 and <TEX>$37^{\circ}C$</TEX> respectively and the <TEX>$CO_2$</TEX> concentration was 1, 5, 10 and 15%. We confirmed that sialidase activity was the lowest in biphasic culture that was shifted from normal culture condition to 1% of <TEX>$CO_2$</TEX> and <TEX>$33^{\circ}C$</TEX> on day 5. However, the temperature and concentration of <TEX>$CO_2$</TEX> have little effect on activity of <TEX>${\alpha}2,3$</TEX>-sialyltransferase. Also, sialic acid contents were enhanced 1.13-fold higher than that in control culture. In conclusion, Biphasic cultivation in CHO cells led to inhibition of sialidase activity and increases of sialylated glycan.

Biphasic cultivation strategy to avoid Epo-Fc aggregation and optimize protein expression

In biphasic cultivations, the culture conditions are initially kept at an optimum for rapid cell growth and biomass accumulation. In the second phase, the culture is shifted to conditions ensuring maximum specific protein production and the protein quality required. The influence of specific culture parameters is cell line dependent and their impact on product quality needs to be investigated. In this study, a biphasic cultivation strategy for a Chinese hamster ovary (CHO) cell line expressing an erythropoietin fusion protein (Epo-Fc) was developed. Cultures were run in batch mode and after an initial growth phase, cultivation temperature and pH were shifted. Applying a DoE (Design of Experiments) approach, a fractional factorial design was used to systematically evaluate the influence of cultivation temperature and pH as well as their synergistic effect on cell growth as well as on recombinant protein production and aggregation. All three responses were influenced by the cultivation temperature. Additionally, an interaction between pH and temperature was found to be related to protein aggregation. Compared with the initial standard conditions of 37°C and pH 7.05, a parameter shift to low temperature and acidic pH resulted in a decrease in the aggregate fraction from 75% to less than 1%. Furthermore, the synergistic effect of temperature and pH substantially lowered the cell-specific rates of glucose and glutamine consumption as well as lactate and ammonium production. The optimized culture conditions also led to an increase of the cell-specific rates of recombinant Epo-Fc production, thus resulting in a more economic bioprocess.

Production of a bioherbicide agent in liquid and solid medium and in a biphasic cultivation system

The use of fungi in weeds control programs depends upon the conidia production in large scale. Therefore, this study aimed to evaluate liquid and solid culture media and the cultivation by biphasic system for the conidia production of Bipolaris euphorbiae Muchovej &amp; Carvalho a specific pathogen of Euphorbia heterophylla. The liquid media were obtained from agro-industrial waste or by-products, and the solid media were prepared with mixtures of grains and grain derivatives. The liquid medium made with sugar cane molasses stood out from the others because it provided great sporulation (23 x 10(4) conidia mL-1 of medium), conidial viability (99.7%), and formation of mycelial fungal biomass (1.26 g 100 mL-1 of medium). On solid media conidial production was markedly higher than in liquid media, especially the medium composed by a blend of sorghum grain (40%) and soybean hulls (60%) where the fungus produced 2.3 x 10(7) conidia g-1 of medium. The cultivation of B. euphorbiae in biphasic system not promoted a significant increase in the production of conidia. The solid media were more effective for the mass production of fungus and mixtures of grains and derivatives were effective for increasing conidia production.

Phototrophic Growth and Accumulation of Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by Purple Nonsulfur Bacterium Rhodopseudomonas palustris SP5212

The ability of the phototrophic bacterium Rhodopseudomonas palustris SP5212 to produce polyhydroxyalkanoates (PHAs), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] in particular was, assessed in acetate medium supplemented with hydroxybutyrate and valerate as cosubstrates. The isolate accumulated the polymer accounting for some 49.06% and 30% of cell dry weight when grown in hydroxybutyrate and valerate, respectively. PHA accumulation as well as 3HV monomer incorporation (30 mol%) was maximum at 0.1% hydroxybutyrate, while valerate at 0.1% and 0.3% was suitable for total polymer accumulation and 3HV monomer incorporation, respectively. Cosupplementation of hydroxybutyrate and valerate in the ratio of 3 : 1 led to the accumulation of PHA accounting for 54% of cell dry weight, which contained more than 50 mol% of 3HV monomer. Moreover, the biphasic cultivation conditions with hydroxybutyrate as cosubstrate have improved the quality as well as quantity of the accumulated copolymer significantly.

Increasing the productivity of TNFR-Fc in GS-CHO cells at reduced culture temperatures

In an effort to improve TNFR-Fc production in GS-CHO cells, batch cultures were performed to investigate the effects of low culture temperature on TNFR-Fc production. It was observed that low culture temperatures resulted in cell cycle arrest in the G1 phase, led to suppressed cell growth, and prolonged the culture period. Although the highest TNFR-Fc concentration was achieved with a culture temperature of 32°C at 247.4 mg/L, the maximum q TNFR-Fc of 15.7 pg/cells/day was achieved at 30°C. Because the inhibition effect on cell growth at 30°C compromised its beneficial effects specifically to TNFR-Fc productivity, TNFR-Fc concentration at this temperature was not significantly increased. Furthermore, the increase in productivity of specific TNFR-Fc at low culture temperatures was also found to be due to an increase in the transcriptional level of the TNFR-Fc gene, determined by RT-PCR analysis. In addition, low culture temperatures had no significant effect on the degree of sialylation of TNFR-Fc. Taken together; a biphasic cultivation process developed in a fed-batch mode with a low temperature-production phase enhanced TNFR-Fc production by GS-CHO cells and therefore offers major potential for bioprocess optimization.

Process parameter shifting: Part II. Biphasic cultivation—A tool for enhancing the volumetric productivity of batch processes using Epo‐Fc expressing CHO cells

Regulation of cell growth and protein expression potentially results in a sustainable enhancement of the volumetric productivity in a fermentation process. Following a biphasic cultivation strategy the process initially passes through a cell proliferation phase to generate a sufficiently high viable cell mass. In the subsequent production phase cells are maintained viable and productive without significant cell proliferation leading to increased viable cell days and product yields. In a previous work we have shown that the well directed alteration of the process environment based on process parameter shifting is a promising tool to regulate cell growth and protein expression. In continuation of this work we investigated process parameters which have been identified to affect cell proliferation in favor of an increased specific productivity and total product yield in a series of biphasic batch cultivation experiments. In most of these processes the integral of viable cells and the specific productivity were increased leading to a significant improvement of both final product concentration and volumetric productivity. In addition, combined parameter shifts (pH 6.90/30 degrees C and pH 6.90/33 degrees C) exerted a synergistic effect on product quality. The loss of product sialylation which occurred at reduced temperatures was prevented by simultaneously reducing the external pH. In conclusion, biphasic cultivation based on combined shifting of process parameters is a suitable tool for controlling cell proliferation and protein expression of mammalian cells in a batch bioreactor leading to enhanced volumetric productivities and therefore offers an enormous potential for bioprocess optimization.

Process parameter shifting: Part I. Effect of DOT, pH, and temperature on the performance of Epo‐Fc expressing CHO cells cultivated in controlled batch bioreactors

The impact of process environment changes on process performance is one of the most crucial process safety issues when cultivating mammalian cells in a bioreactor. In contrast, directed shifting of process parameters can also be used as an optimization tool providing higher cell and product yields. Compared to other strategies that also aim on the regulation of cell growth and protein expression process parameter shifts can be easily performed without reagent addition or even genetic modification of the host cell line. However, a successful application of changing process conditions implies a profound understanding of the provoked physiological changes within the cells. In a systematic approach we varied the dissolved oxygen tension (DOT), pH, and temperature of CHO cultures in controlled bioreactors and investigated the impact on growth, productivity, metabolism, product quality and cell cycle distribution using a recombinant CHO cell line expressing the highly glycosylated fusion protein Epo-Fc. We found the reduction of cultivation temperature and the reduction of (external) pH to exert the most significant effects on process performance by mainly reducing cell growth and metabolism. With respect to the cell line used we identified a set of parameters capable of affecting cell proliferation in favor of an increased specific productivity and total product yield. The well directed alteration of the process environment has emerged as a tool adequate for further process optimization applying a biphasic cultivation strategy.

A biphasic chamber system for maintaining polarity of differentiation of cultured respiratory tract epithelial cells.

A simple, disposable, biphasic cultivation chamber has been developed for respiratory tract epithelial cells. This chamber, the Whitcutt chamber, contains a movable, transparent, permeable gelatin membrane that can be employed either submerged in the culture medium, thereby feeding the cells by the traditional immersion method, or raised to the surface of the culture medium, to bring the apical surfaces of the cells into contact with air and provide nutrients only from below (basal feeding). The effects of biphasic cultivation on the growth and differentiation of respiratory tract epithelial cells from different sources have been studied in Whitcutt chambers. Primary hamster tracheal epithelial (HTE) cells grown to confluence with basal feeding developed a ciliated columnar morphology, with differentiated features (cilia and mucous granules) located in the apical region of the epithelial layer. These cells secreted mucinlike molecules from the apical surface (i.e. the surface in contact with air). Although the apical localization of differentiation features was greater, mucous cell differentiation achieved by basal feeding was quantitatively not greater than that achieved by continuous immersion feeding. Similarly, basal feeding did not alter the degree of epithelial cell differentiation in cultures derived from rat, rabbit, and monkey tracheas or from human bronchial and nasal tissues. In contrast, the differentiation of guinea pig tracheal epithelial cells in culture was significantly influenced by the feeding method employed. When fed basally, guinea pig tracheal epithelial cell cultures expressed various mucociliary functions with resemblance to mucociliary layers in vivo, whereas constantly immersed cultures seemed stratified and squamous. These results suggest that, at least for guinea pigs, the combination of feeding methods provided by the Whitcutt chamber can be used to achieve differentiated cultures of tracheal epithelial cells with a polarity of differentiation that is similar to that observed in intact airways in vivo.