Growth Of Suspension Cultures Research Articles

Establishment and optimization of cell growth in suspension culture of Papaver bracteatum: a biotechnology approach for thebaine production

Iranian poppy (Papaver bracteatum) is an important medicinal plant that is the main source of the opium alkaloids codeine, morphine, and thebaine. To establish an efficient protocol for cell suspension culture and growth, the effects of different plant growth regulators (2,4-D, NAA, BAP, and kinetin) on callus induction and cell suspension culture of Iranian poppy were evaluated. The maximum percentage of callus induction (86.67%) and fresh weight of callus were obtained in MS medium supplemented with 1 or 2 mg/L 2,4-D, 0.1 or 0.2 mg/L kinetin and 15 mg/L ascorbic acid. The addition of 15 mg/L ascorbic acid to the medium was effective in reducing browning of explants and calli. In cell suspension cultures, MS medium supplemented with 1 mg/L NAA, 1 mg/L BAP, and 15 mg/L ascorbic acid gave maximum cell growth and proliferation compared to other treatments evaluated. Among the auxins, NAA was more effective than 2,4-D for growth of P. bracteatum cell suspension cultures. However, there was no statistically significant difference between kinetin and BAP. The thebaine yield of P. bracteatum cell suspension at 12 days after culture was 4.67 µg/g dry weight, which was significantly lower than that at 16 days (10.76 µg/g dry weight).

Identification of a Novel Protein-Coding Gene (TIHL) and Its Functional Relevance in Myeloid Cells.

AbstractAbstract 2333The gene locus BX648577 (FLJ27352/hypothetical LOC145788) was recently identified as part of a gene fusion with Class II Transactivator (CIITA) in Hodgkin's lymphoma using whole-transcriptome paired-end sequencing (Steidl C. et al., Nature 2011). While CIITA has been extensively studied, no function is known for BX648577. We therefore sought to study its expression and biological function in normal and malignant hematopoietic cells, including its effects on cellular proliferation, clonogenicity and cell death. Here, we report the endogenous expression of functional mRNA and protein encoded by the BX648577 locus, thereby establishing it as a novel protein-coding gene, which we named TIHL(translocated in Hodgkin's lymphoma).Initial comparative analysis of the 13–16kDa predicted BX648577 (TIHL) protein sequences in various species revealed high evolutionary conservation (≥54%), including invertebrates (e.g. Saccoglossus kowalevskii). We generated a TIHL(BX648577) -specific antibody and identified endogenous protein expression of TIHL by western blot analysis in several cell lines, including 293T cells, and myeloid NB4 and KG1a cells. Expression analysis was performed in several leukemia and lymphoma cell lines by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). And we found that TIHL mRNA was widely and variably expressed, with particularly high expression in NB4 and THP1 myeloid leukemia cells, 5 fold (p=2.02e-6) and 9 fold (p=6.96e-7) higher, respectively, when compared to healthy donor peripheral blood CD14+ monocytes. In addition, TIHL mRNA expression was detectable in sorted, primary human and murine bone marrow derived hematopoietic stem cells (Lin−CD34+CD38− (human) or Lin−c-Kit+Sca-1+ (mouse)) and progenitor cells (Lin−CD34+CD38+ (human) or Lin−c-Kit+Sca-1−(mouse)), as well as human healthy donor mature peripheral blood mononuclear cells with the most prominent expression in Glycophorin A and CD56 positive cells by qRT-PCR.Utilizing primary immature murine bone marrow cells (sorted Lin−c-Kit+ cells) as well as acute myeloid leukemia cell lines (NB4, KG1a); we assessed the biological consequences of modulating TIHL expression. Knockdown of TIHL by two independent shRNAs with knockdown efficiencies of 77% and 50% respectively, led to significantly decreased leukemic cell growth in suspension culture. We additionally observed decreased clonogenic capacity in both cell lines when the cells were transduced with either sh#1 or sh#2, with 76% (p =1.19e-3) and 77% (p=8.36e-4) inhibition in clonogenicity in NB4 cells when compared to cells transduced with a non-silencing control vector. Similarly, we observed 67% (p=3.28e-3) and 72% (p=4.9e-4) inhibition of clonogenicity in KG1a cells. In addition, knockdown of TIHL led to a 7–14 fold increase in specific apoptosis. We did not find changes indicative of differentiation when we analyzed cellular morphology and surface protein expression. We cloned the full length cDNA of TIHL of human and mouse into a lentiviral expression vector. While ectopic expression of human or murine TIHL in sorted Lin−c-Kit+cells did not lead to a change in clonogenicity when compared to the empty control vector, we found a 1.5 fold increase in colony forming capacity (p=0.0161), and a 2 fold increase in cell growth (p<0.05) in NB4 cells upon TIHL overexpression.Finally, in silico domain analysis suggests that TIHL may function as an adaptor protein and may be involved in facilitating previously established signal transduction pathways.In summary, we here identify TIHL (former gene locus BX648577) as a novel protein-coding gene. We show that TIHL is expressed at the mRNA and protein level with wide expression in both leukemia cell lines and normal hematopoietic cells, including stem cells. We demonstrate that TIHL is functionally relevant and that it is involved in the regulation of growth in myeloid cells. Further studies are warranted to better understand the biological role of TIHL, to examine its precise mechanisms of action, and to delineate the biological pathways implicated in its function. Disclosures:No relevant conflicts of interest to declare.

Establishment and characterization of Stevia rebaudiana (Bertoni) cell suspension culture: an in vitro approach for production of stevioside

A protocol has been standardized for establishment and characterization of cell suspension cultures of Stevia rebaudiana in shake flasks, as a strategy to obtain an in vitro stevioside producing cell line. The effect of growth regulators, inoculum density and various concentrations of macro salts have been analyzed, to optimize the biomass growth. Dynamics of stevioside production has been investigated with culture growth in liquid suspensions. The callus used for this purpose was obtained from leaves of 15-day-old in vitro propagated plantlets, on MS medium fortified with benzyl aminopurine (8.9 μM) and naphthalene acetic acid (10.7 μM). The optimal conditions for biomass growth in suspension cultures were found to be 10 g l−1 of inoculum density on fresh weight basis in full strength MS liquid basal medium of initial pH 5.8, augmented with 2,4-dichlorophenoxy acetic acid (0.27 μM), benzyl aminopurine (0.27 μM) and ascorbic acid (0.06 μM), 1.0× NH4NO3 (24.7 mM), 3.0× KNO3 (56.4 mM), 3.0× MgSO4 (4.5 mM) and 3.0× KH2PO4 (3.75 mM), in 150 ml Erlenmeyer flask with 50 ml media and incubated in dark at 110 rpm. The growth kinetics of the cell suspension culture has shown a maximum specific cell growth rate of 3.26 day−1, doubling time of 26.35 h and cell viability of 75 %, respectively. Stevioside content in cell suspension was high during exponential growth phase and decreased subsequently at the stationary phase. The results of present study are useful to scale-up process and augment the S. rebaudiana biological research.

A NanoDrop-based method for rapid determination of viability decline in suspension cultures of animal cells

We describe a rapid method for monitoring the cell growth and decline phases in suspension cultures of animal cells. During the cell growth phase, ultraviolet (UV)-absorbing components in the medium are consumed, but at later times as cells begin to die, UV-absorbing molecules such as proteins are released into the medium. Measuring the absorbance at 280nm (A280) with a NanoDrop spectrophotometer, an inverse correlation between the onset of the cell decline phase and A280 was observed. This simple method can be applied to quickly determine the beginning of the decline phase of cultures of mammalian and insect cells in suspension.

Reuteran and levan as carbohydrate sinks in transgenic sugarcane

The present study reports the effect of high molecular weight bacterial fructan (levan) and glucan (reuteran) on growth and carbohydrate partitioning in transgenic sugarcane plants. These biopolymers are products of bacterial glycosyltransferases, enzymes that catalyze the polymerization of glucose or fructose residues from sucrose. Constructs, targeted to different subcellular compartments (cell wall and cytosol) and driven by the Cauliflower mosaic virus-35S: maize-ubiquitin promoter, were introduced into sugarcane by biolistic transformation. Polysaccharide accumulation severely affected growth of callus suspension cultures. Regeneration of embryonic callus tissue into plants proved problematic for cell wall-targeted lines. When targeted to the cytosol, only plants with relative low levels of biopolymer accumulation survived. In internodal stalk tissue that accumulate reuteran (max 0.03mg/g FW), sucrose content (ca 60mg/g FW) was not affected, while starch content (<0.4mg/g FW) was increased up to four times. Total carbohydrate content was not significantly altered. On the other hand, starch and sucrose levels were significantly reduced in plants accumulating levan (max 0.01mg/g FW). Heterologous expression resulted in a reduction in total carbohydrate assimilation rather than a simple diversion by competition for substrate.

Effects of polysaccharide elicitors from endophytic Fusarium oxysporium Dzf17 on growth and diosgenin production in cell suspension culture of Dioscorea zingiberensis.

Three polysaccharides, namely exopolysaccharide (EPS), water-extracted mycelial polysaccharide (WPS) and sodium hydroxide-extracted mycelial polysaccharide (SPS), were prepared from the endophytic fungus Fusarium oxysporium Dzf17 isolated from the rhizomes of Dioscorea zingiberensis. The effects of the time of addition and polysaccharide concentration on the growth and diosgenin accumulation in cell suspension culture of D. zingiberensis were studied. Among them, WPS was found to be the most effective polysaccharide. When WPS was added to the medium at 20 mg/L on the 25th day of culture, the cell dry weight was increased 1.34-fold, diosgenin content 2.85-fold, and diosgenin yield 3.83-fold in comparison to those of control. EPS and SPS showed moderate and relatively weak enhancement effects on cell growth and diosgenin accumulation, respectively. The dynamics of cell growth and diosgenin accumulation when WPS was added to the medium at 20 mg/L on the 25th day of culture were investigated, and results showed that dry weight of cells reached a maximum value on day 30 but the maximum diosgenin content was achieved on day 31.

Development of a measles vaccine production process in MRC-5 cells grown on Cytodex1 microcarriers and in a stirred bioreactor

Measles vaccination remains the most efficient way to control the spread of the virus. This work focuses on the production of a measles vaccine using stirred conditions as an advanced option for process scale up. Non-porous Cytodex 1 microcarriers were used to support MRC-5 cell growth in suspension cultures. Virus replication was first optimized in spinner flasks, and the effects of various operational parameters were investigated. Cell infection with AIK-C measles strain at an MOI (multiplicity of infection) of 0.005, without glucose regulation and in M199 medium, resulted in a virus titer of 10⁶·²⁵ TCID₅₀ (median tissue culture infective dose)/ml. To optimize the production process in a 7-l bioreactor, we carried out various perfused cultures using minimum essential medium (MEM) + 5% FCS diluted with phosphate-buffered saline (PBS). We achieved a high cell density level (4.1 × 10⁶ cells/ml) with an efficient use of the medium when MEM + 5% FCS diluted with PBS at 25% was used during the cell amplification step. Optimization of measles production in MRC-5 cells grown on Cytodex 1 beads in a 7-l bioreactor showed that perfusion was the most efficient when compared to repeated-batch culture. Perfusion at a rate of 0.25 V (reactor volume)/day showed the highest specific productivity (1.6 IVP [infectious virus particle] cell⁻¹ day⁻¹). Testing of several stabilizers containing pharmaceutically improved components such as sugars, amino acids, and charged ions showed that the formulation composed of sucrose and MgCl₂, led to the maintenance of the infectivity of the AIK-C measles virus strain to a significant level, when stored at +28 °C, +4 °C and -60 °C.

Induced accumulation of 20-hydroxyecdysone in cell suspension cultures of Vitex glabrata R.Br.

This study describes the effects of culture medium, culture temperature, sucrose concentration and cholesterol feeding on cell growth and 20-hydroxyecdysone production in suspension cultures of Vitex glabrata , an important medicinal plant in Thailand. Cell growth and 20-hydroxyecdysone production were not significantly different when cells were cultivated on B5 or half-strength MS medium. However, cultivation of V. glabrata cell cultures at 25°C yielded 1.06- and 1.09-fold higher values of cell growth and 20-hydroxyecdysone content, respectively than those at 30°C. Sucrose at 30 and 40 g/L favors the production of 20-hydroxyecdysone in suspension cultures of V. glabrata . Feeding of cholesterol at 5 mg/L, as precursor for biosynthesis of 20-hydroxyecdysone, yielded 1.11-fold higher accumulation of 20-hydroxyecdysone than the control cells. Increasing of cholesterol to 10 mg/L resulted in decreased production of 20-hydroxyecdysone. Key words : Vitex glabrata, 20-hydroxyecdysone, suspension culture, cholesterol.

Significant Enhancement of Triterpenoids Production by &lt;i&gt;Phomopsis&lt;/i&gt; Elicitors in Cell Suspension Cultures of &lt;i&gt;Betula Platyphylla Suk&lt;/i&gt;.

58 fungal elicitors prepared from the endophytic fungi isolated from inner bark of B. platyphylla Suk. were examined to determine their effects on the growth and triterpenoid production in suspension cultures of Betula platyphylla Suk. cells. The results showed that different fungal elicitors could cause diversely stimulating effects. Among the fungal elicitors tested, BE58 stimulated the highest triterpenoids yield with 15.90 mg•L-1 and 183.72% higher than the control. The experiment also affirmed from mRNA level that triterpenoid was indeed accumulated in suspension culture of birch cells treated by BE58 fungal elicitor. Meantime BE58 was identified as Phomopsis from the morphological and molecular level.

Recombinant glycoprotein E produced in mammalian cells in large-scale as an antigen for varicella-zoster-virus serology

A recombinant glycoprotein E (gE) from varicella-zoster virus (VZV) was generated and produced in Chinese Hamster Ovary (CHO) cells, in the development of a specific antigen for analysis of IgG antibodies to VZV. Several stable gE-secreting clones were established and one clone was adapted to growth in serum-free suspension culture. When the cells were cultured in a perfusion bioreactor, gE was secreted into the medium, from where it could be easily purified. The recombinant gE was then evaluated as a serological antigen in ELISA. When compared to a conventional whole virus antigen, the VZV gE showed similar results in ELISA-based seroprevalence studies of 854 samples derived from blood donors, students, ischemic stroke patients and their controls, including samples with border-line results in previous analyses. Eight samples (0.9%) were discordant, all being IgG-negative by the VZV gE ELISA and positive by the whole virus ELISA. The sensitivity and specificity of the VZV gE ELISA were 99.9% and 100%, respectively, compared to 100% and 88.9% for the VZV whole virus ELISA. The elderly subjects showed similar reactivities to both antigens, while VZV gE gave lower signals in the younger cohorts, suggesting that antibodies to gE may increase with age. It was concluded that the recombinant VZV gE from CHO cells was suitable as a serological antigen for the detection of IgG antibodies specific for VZV.

Growth and Alkaloid Accumulation in Cell Suspension Cultures of Ailanthus Altissima

Growth and Alkaloid Accumulation in Cell Suspension Cultures of <u>Ailanthus Altissima</u>

Membrane-associated Ubiquitin Ligase Complex Containing gp78 Mediates Sterol-accelerated Degradation of 3-Hydroxy-3-methylglutaryl-coenzyme A Reductase

The endoplasmic reticulum (ER)-associated degradation (ERAD) pathway in the yeast Saccharomyces cerevisiae is mediated by two membrane-bound ubiquitin ligases, Doa10 and Hrd1. These enzymes are found in distinct multiprotein complexes that allow them to recognize and target a variety of substrates for proteasomal degradation. Although multiprotein complexes containing mammalian ERAD ubiquitin ligases likely exist, they have yet to be identified and characterized in detail. Here, we identify two ER membrane proteins, SPFH2 and TMUB1, as associated proteins of mammalian gp78, a membrane-bound ubiquitin ligase that bears significant sequence homology with mammalian Hrd1 and mediates sterol-accelerated ERAD of the cholesterol biosynthetic enzyme HMG-CoA reductase. Co-immunoprecipitation studies indicate that TMUB1 bridges SPFH2 to gp78 in ER membranes. The functional significance of these interactions is revealed by the observation that RNA interference (RNAi)-mediated knockdown of SPFH2 and TMUB1 blunts both the sterol-induced ubiquitination and degradation of endogenous reductase in HEK-293 cells. These studies mark the initial steps in the characterization of the mammalian gp78 ubiquitin ligase complex, the further elucidation of which may yield important insights into mechanisms underlying gp78-mediated ERAD.

Overexpression of survivin and cyclin D1 in CHO cells confers apoptosis resistance and enhances growth in serum-free suspension culture

To optimize Chinese hamster ovary (CHO) cell culture to recombinant protein therapeutic production, we stably overexpressed survivin and cyclin D1 in three CHO DG44-derived cell lines. The modifications conferred increases of 56-94% in S-phase fractions and decreases of 33-43% in early-stage apoptosis fractions. Clone 6.3, which expressed the highest levels of survivin and cyclin D1, reached significantly greater cell densities in suspension (2.7 × 10(6) cells/ml) following serum deprivation. Nude mice inoculated with the modified cells showed no tumorigenesis suggesting that the CHO DG44-derived cell lines are viable candidates for biopharmaceutical production.

Nitrosyl ethylenediaminetetraacetate ruthenium(II) complex promotes cellular growth and could be used as nitric oxide donor in plants

Over recent years nitric oxide (NO) not only has appeared as an important endogenous signaling molecule in plants and as a mediator in many developmental and physiological processes, but has also received recognition as a plant hormone. The impressive recent achievements in elucidating the role of NO in plants have come about by the application of NO donors. The aim herein was to study the effects of the different NO donors, sodium nitroprusside (SNP) and the nitrosyl ethylenediaminetetraacetate ruthenium(II) ([Ru(NO)(Hedta)]) complex on cellular growth in embryogenic suspension cultures of Araucaria angustifolia. Appraisal of our data revealed that [Ru(NO)(Hedta)] stimulated about 60% of cellular growth in embryogenic suspension cultures of A. angustifolia, with results similar to those observed with the SNP donor. Nevertheless, application of the NO scavenger PTIO (2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide) inhibited this cellular growth in both. Cellular growth was correlated with an increase in endogenous NO levels after 21 days of culture, especially in treatments with NO donors. Our results demonstrated that the [Ru(NO)Hedta] complex could possibly be used as a NO donor in plants.

Development of embryo-like structures in the suspension cultures of flax coincides with secretion of chitinase-like proteins

Flax suspension cultures have been established from the callus induced from the cotyledons, hypocotyls, and immature zygotic embryos (iZE). The growth of flax suspension culture (expressed as a sedimented cell volume) was compared in both conditioned (by liquid from embryogenic Pinus nigra suspension culture) and non-conditioned media. Conditioning of media significantly increased the growth of the cell lines of hypocotyl and iZE origin; however, it had no promotive effect on embryogenic response of these flax liquid cultures. Formation of embryo-like structures (ELS), confirmed also histologically, has only been found in the cell line derived from iZE and cultivated in non-conditioned MS medium supplemented with 1 mg l−1 2,4-D. The process of ELS formation in this cell line was accompanied by the expression of the protein(s) with chitinolytic activity and molecular weight approximately of 25 kDa. The relationship between the formation of ELS and secretion of chitinase(s) is discussed.

Interaction of a living system with an electromagnetic field

Irradiation of the inoculum with 50 Hz frequency electromagnetic field for 3 h practically did not influence the growth of suspension culture of one-celled green algae Dunaliella tertiolecta and Tetraselmis viridis. The irradiation for 4 h led to approximately 30% reduction of growth. After 6-hour irradiation the growth of Dunaliella tertiolecta was reduced by 50% and the growth of Tetraselmis viridis was reduced by 70%. The irradiation of hydras for 3 h showed no visible effect. The irradiation for 4 h stimulated a little the growth and regeneration of injured hydras. The effect of 24 h irradiation resulted in the shortening of lifetime of uninjured and injured hydras. The effect of regeneration was not observed.

Differential effects of overexpression of ERα and ERβ in MCF10A immortalised, non-transformed human breast epithelial cells.

Cellular effects of oestrogen are mediated by two intracellular receptors ERα and ERβ. However, to compare responses mediated through these two receptors, experimental models are needed where ERα and ERβ are individually stably overexpressed in the same cell type. We compared the effects of stable overexpression of ERα and ERβ in the MCF10A cell line, which is an immortalised but non-transformed breast epithelial cell line without high endogenous ER expression. Clones of MCF10A cells were characterised which stably overexpressed ERα (10A-ERα2, 10A-ERα13) or which stably overexpressed ERβ (10A-ERβ12, 10A-ERβ15). Overexpression of either ERα or ERβ allowed induction of an oestrogen-regulated ERE-LUC reporter gene by oestradiol which was not found in the untransfected cells. Oestradiol also increased proliferation of 10A-ERα13 and 10A-ERβ12 cells, but not untransfected cells, by 1.3-fold over 7 days. The phytoestrogen, genistein, which is reported to bind more strongly to ERβ than to ERα, could induce luciferase gene expression from an ERE-LUC reporter gene at concentrations of 10-6 M and 10-5 M but only in the clones overexpressing ERβ and not in those overexpressing ERα. Clone 10A-ERβ12 also yielded growth stimulation with 10-6 M genistein. Finally, the overexpression of ERα, but not ERβ, gave rise to increased growth in semi-solid methocel suspension culture in the presence of 70nM oestradiol, suggesting that overexpression of ERα, but not ERβ, produces characteristics of a transformed phenotype. This provides a model system to compare effects of oestradiol with other oestrogenic ligands in cells stably overexpressing individually ERα or ERβ.

Influence of growth regulators and sucrose concentrations on growth and rosmarinic acid production in calli and suspension cultures of Coleus blumei

Rosmarinic acid, which is reported to have adstringent, antibacterial, antiviral and antioxidant activities, is one of the most prominent secondary compounds in Coleus blumei (Lamiaceae). Rosmarinic acid (RA) production in different hybrids of C. blumei was estimated by HPLC. Conditions for HPLC were as follows: column, 150 × 4.6 mm; solvent system, methanol −0.1% phosphate (45 : 55); flow rate, 0.9 mL/min; detection: 325 nm. Two out of four hybrids of C. blumei (hy1; hy2) contain better rosmarinic acid production (0.9 and 1.0% dry weight, respectively) and the leaves have the highest rosmarinic acid production, followed by stems and roots. The hydroxyphenylpyruvate reductase (HPPR) gene expression levels were analysed by semi-quantitative RT-PCR. Hy3 shows highest level of HPPR gene expression out of four hybrids on genotype-specific patterns, and stems represent the highest level of HPPR gene expression among leaves, roots and stems. This was probably a result of the fact that the RA biosynthetic pathway was regulated by interactions of several enzymes necessary for biosynthesis. The explants from the hy1 leaves were used in subsequent studies on the effect of different growth regulators (2.0 mg L−1 6-benzyl-aminopurine (6-BA), different 2,4-dichlorophenxyaretic acid (2,4-D) and α-naphthaleneacetic (NAA) concentrations) and sucrose contents (1, 2, 3, 4, 5 or 6%) on culture growth and rosmarinic acid accumulation. On the effect of different growth regulators, the best result is obtained when the B5-medium supplemented is with 2.0 mg L−1 6-BA, 0.5 mg L−1 NAA, 0.8 mg L−1 2,4-D and 2% sugar, and solidified with 0.8% agar. In this case, both growth index and rosmarinic acid accumulation reach a maximum, which is 49.7 and 25.3% (dry weight), respectively. The optimal medium for suspension culture growth contains 2.0 mg L−1 6-BA, 0.5 mg L−1 NAA, 0.8 mg L−1 2,4-D, 600 mg L−1 inositel and 2% sugar, and the rosmarinic acid production is 1.7% (dry weight). The rosmarinic acid accumulation is enhanced by the sucrose concentrations. When the sucrose content is 6%, the calli and cell culture accumulate 33.7% (dry weight) and 10.1% (dry weight) rosmarinic acid, respectively. Higher sucrose content (≥5%) has a negative influence on culture growth. B5-medium supplemented with 2.0 mg L−1 6-BA, 0.5 mg L−1 NAA, 0.8 mg L−1 2,4-D, 4% sugar and 0.8% agar is best for both culture growth and rosmarinic acid accumulation. On the basis of the above results, we can establish a two-phase culture system by calli of C. blumei for the biosynthesis of RA. In the growth phase, the calli grow well, but with lower RA production in a modified B5-1A medium with 2% sucrose. The calli are then subcultured to B5-1A medium with 6% sucrose where the calli start to accumulate RA, but grow slowly.

Influence of Auxins and Sucrose in Monoterpenoid Oxindole Alkaloid Production by Uncaria tomentosa Cell Suspension Cultures

Growth and alkaloid production in Uncaria tomentosa cell suspension cultures were studied in Murashige and Skoog medium supplemented with 10 microM 2,4-dichlorophenoxyacetic acid, 10 microM kinetin, and 58 mM sucrose for maintenance and with 10 microM indole-3-acetic acid, 10 microM kinetin, and 58 mM sucrose for production. A U. tomentosa pale Uth-3 cell line, cultured in the production medium, showed a reduced lag phase and a specific growth rate (mu) of 0.27 day(-1), while cells growing in the maintenance medium showed mu = 0.20 day(-1). U. tomentosa cells growing in the production medium produced monoterpenoid oxindole alkaloids (MOA) in amounts of 10.2 +/- 1.6 microg g(-1) dry weight (DW). The chemical profile of MOA produced by in vitro cell cultures was similar to that found in the plant. After 10 subcultures, maximum MOA production decreased to 2.0 +/- 0.7 microg g(-1) DW, while tryptamine alkaloids (TA) were produced with a maximum of 6.2 +/- 0.4 microg g(-1) DW. The increase of initial sucrose concentration up to 145 mM in the production medium enhanced the cell biomass by 3.2-fold (from 10.2 +/- 0.1 to 32.8 +/- 1.1 g DW L(-1)), reduced mu from 0.27 to 0.23 day(-1), and provoked a substantial accumulation of TA (23.1 +/- 4.7 microg g(-1) DW). A high sucrose concentration stimulated MOA production in the maintenance medium (2.7 +/- 0.5 microg g(-1) DW), even in the presence of 2,4-dichlorophenoxyacetic acid.

Competition strategies for the incubation of white rot fungi under non-sterile conditions

White rot fungi is capable of oxidizing many persistent organic pollutants including dyes. The application of the fungi has been limited for wastewater treatment under non-sterile conditions due to the contamination by bacteria and other micro-organisms. We developed a treatment approach by using immobilized white rot fungi Phanerochaete chrysosporium to degrade reactive dye K-2BP under non-sterile condition. Four different inert carriers were tested for immobilization of the white rot fungi with orthogonal experiments in comparison with suspension culture. The activity of manganese peroxidase (MnP) was used for the evaluation of oxidization performance in order to understand whether contamination of bacteria and other micro-organisms was suppressed. Under non-sterile conditions, the immobilized fungal cultures successfully restrained the growth of microzymes, coccies, and bacillus but suspension culture was highly contaminated with poor MnP activity. Under non-sterile conditions, higher MnP enzymatic activity (690 U/L vs. 125 U/L), higher decolorization efficiency (93.5% vs. 15%) and shorter reaction period (3 days vs. 6 days) were achieved in immobilized cultures in comparison with suspension culture. With the immobilized fungal cultures, no difference was observed under non-sterile and sterile conditions for the degradation of reactive dye K-2BP.