Statistical medium optimization and biodegradative capacity of <Emphasis Type="Italic">Ralstonia eutropha</Emphasis> toward <Emphasis Type="Italic">p</Emphasis>-nitrophenol

The effect of p-nitrophenol (PNP) concentration with or without glucose and yeast extract on the growth and biodegradative capacity of Ralstonia eutropha was examined. The chemical constituents of the culture medium were modeled using a response surface methodology. The experiments were performed according to the central composite design arrangement considering PNP, glucose and yeast extract as the selected variables whose influences on the degradation was evaluated (shaking in reciprocal mode, temperature of 30°C, pH 7 and test time of about 9 h). Quadratic polynomial regression equations were used to quantitatively explain variations between and within the models (responses: the biodegradation capacity and the biomass formation). The coefficient of determination was high (R _adjusted² = 0.9783), indicating the constructed polynomial model for PNP biodegradative capacity explains the variation between the regressors fairly well. A PNP removal efficiency of 74.5% occurred within 9 h (15 mg/L as the initial concentration of PNP with use of yeast extract at 0.5 g/L).     

Utilization of dibenzothiophene as sulfur source by <Emphasis Type="Italic">Microbacterium</Emphasis> sp. <Emphasis Type="Italic">NISOC-06</Emphasis>

Oil-polluted soils were sampled from National Iranian South Oil Company (NISOC) for isolation and screening of C–S and not C–C targeted Dibenzothiophene (DBT) degrading microorganisms. Microbacterium sp. NISOC-06, a C–S targeted DBT degrading bacterium, was selected and its desulfurization ability was studied in aqueous phase and water-gasoline biphasic systems. The 16srRNA gene was amplified using universal eubacteria-specific primers, PCR product was sequenced and the sequence of nearly 1,500 bp 16srDNA was studied. Based on Gas Chromatography results Microbacterium sp. NISOC-06 utilized 94.8% of 1 mM DBT during the 2 weeks of incubation. UV Spectrophotometry and biomass production measurements showed that the Microbacterium sp. NISOC-06 was not able to utilize DBT as a carbon source. There was no accumulation of phenolic compounds as Gibb’s assay showed. Biomass production in a biphasic system for which DBT-enriched gasoline was used as the sulfur source indicated the capability of Microbacterium sp. NISOC-06 to desulfurize gasoline.     

DNA plasmid production in different host strains of <Emphasis Type="Italic">Escherichia coli</Emphasis>

We compared plasmid DNA production in 13 strains of Escherichia coli in shake flasks using media containing glucose or glycerol. DNA yield from either carbon source showed small correlation with maximum growth rate. Three strains, SCS1-L, BL21 and MC4100, were selected for a controlled exponential fed-batch process at a growth rate of 0.14 h⁻¹ to an optical density of about 70, followed by a four-hour heat treatment. Prior to heat treatment, SCS1-L generated 15.4 mg DNA/g, BL21 generated 11.0 mg DNA/g and MC4100 generated 7.9 mg DNA/g, while after heat treatment the strains attained DNA yields, respectively, of 18.0, 15.0 and 6.8 mg/g. The strains also varied in their percentage of supercoiled DNA after heat treatment, with SCS1-L averaging 66% supercoiled, BL21 17% and MC4100 40%. We further investigated the two strains that yielded the highest percentage of supercoiled DNA (SCS1-L and MC4100) at a higher growth rate of 0.28 h⁻¹. At this condition, a slightly lower DNA yield was generated faster, and the percentage of supercoiled DNA increased. Heat treatment improved DNA yield, and surprisingly did so to a greater extent at the higher growth rate. As a consequence of these factors, higher growth rates might be advantageous for DNA production.     

Heterologous expression of <Emphasis Type="Italic">Thermobifida fusca</Emphasis> thermostable alpha-amylase in <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> and its application in boiling stable resistant sago starch preparation

A gene encoding the thermostable α-amylase in Thermobifida fusca NTU22 was amplified by PCR, sequenced, and cloned into Yarrowia lipolytica P01g host strain using the vector pYLSC1 allowing constitutive expression and secretion of the protein. Recombinant expression resulted in high levels of extracellular amylase production, as high as 730 U/l in the Hinton flask culture broth. It is higher than that observed in P. pastoris expression system and E. coli expression system. The purified amylase showed a single band at about 65 kDa by SDS-polyacrylamide gel electrophoresis and this agrees with the predicted size based on the nucleotide sequence. About 70% of the original activity remained after heat treatment at 60°C for 3 h. The optimal pH and temperature of the purified amylase were 7.0 and 60°C, respectively. The purified amylase exhibited a high level of activity with raw sago starch. After 72-h treatment, the DP _w of raw sago starch obviously decreased from 830,945 to 237,092. The boiling stable resistant starch content of the sago starch increased from 8.3 to 18.1%. The starch recovery rate was 71%.     

Cyanuric acid biodegradation by a mixed bacterial culture of <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> and <Emphasis Type="Italic">Acinetobacter</Emphasis> sp. in a packed bed biofilm reactor

Cyanuric acid (1,3,5-triazine-2,4,6-triol [OOOT]) is a common biodegradation byproduct of triazinic herbicides, frequently accumulated in soils or water when supplementary carbon sources are absent. A binary bacterial culture able to degrade OOOT was selected through a continuous selection process accomplished in a chemostat fed with a mineral salt (MS) medium containing cyanuric acid as the sole carbon and nitrogen source. By sequence comparison of their 16S rDNA amplicons, bacterial strains were identified as Agrobacterium tumefaciens, and Acinetobacter sp. When the binary culture immobilized in a packed bed reactor (PBR) was fed with MS medium containing OOOT (50 mg L⁻¹), its removal efficiencies were about 95%; when it was fed with OOOT plus glucose (120 mg L⁻¹) as a supplementary carbon source, its removal efficiencies were closer to 100%. From sessile cells, attached to PBR porous support, or free cells present in the outflowing medium, DNA was extracted and used for Random Amplification of Polymorphic DNA analysis. Electrophoretic patterns obtained were compared to those of pure bacterial strains, a clear predominance of A. tumefaciens in PBR was observed. Although in continuous suspended cell culture, a stable binary community could be maintained, the attachment capability of A. tumefaciens represented a selective advantage over Acinetobacter sp. in the biofilm reactor, favoring its predominance in the porous stone support.     

Biosorption of an Azo Dye by <Emphasis Type="Italic">Aspergillus niger</Emphasis> and <Emphasis Type="Italic">Trichoderma</Emphasis> sp. Fungal Biomasses

Biosorption is an eco-friendly and cost-effective method for treating the dye house effluents. Aspergillus niger and Trichoderma sp. were cultivated in bulk and biomasses used as biosorbents for the biosorption of an azo dye Orange G. Batch biosorption studies were performed for the removal of Orange G from aqueous solutions by varying the parameters like initial aqueous phase pH, biomass dosage, and initial dye concentration. It was found that the maximum biosorption was occurred at pH 2. Experimental data were analyzed by model equations such as Langmuir and Freundlich isotherms, and it was found that both the isotherm models best fitted the adsorption data. The monolayer saturation capacity was 0.48 mg/g for Aspergillus niger and 0.45 mg/g for Trichoderma sp. biomasses. The biosorption kinetic data were tested with pseudo first-order and pseudo second-order rate equations, and it was found that the pseudo second-order model fitted the data well for both the biomasses. The rate constant for the pseudo second-order model was found to be 10–0.8 (g/mg min⁻¹) for Aspergillus niger and 8–0.4 (g/mg min⁻¹) for Trichoderma sp. by varying the initial dye concentrations from 5 to 25 mg/l. It was found that the biomass obtained from Aspergillus niger was a better biosorbent for the biosorption of Orange G dye when compared to Trichoderma sp.     

Overexpression of <Emphasis Type="Italic">odc</Emphasis> (ornithine decarboxylase) in <Emphasis Type="Italic">Datura innoxia</Emphasis> enhances the yield of scopolamine

Scopolamine is widely used for its anticholinergic properties. Because of higher physiological activity and less side effects the world demand of scopolamine is estimated to be ten times greater than other anticholinergic agents, hyoscyamine and atropine. Since natural production is limited, alternatives are required to boost the production. We report the introduction of mouse odc gene of polyamine biosynthesis pathway which is also the primary pathway of tropane alkaloids in Datura innoxia. Polyamines, mainly putrescine, serve as the common metabolite for tropane alkaloids and nicotine. We have overexpressed odc gene to modulate the metabolic flux downstream and eventually achieved higher accumulation of scopolamine in transgenic plants. Among six independent transformed lines one line (O10) produced scopolamine (0.258 μg/g dry weight) almost six times higher than that produced by control plants (0.042 μg/g DW). To our knowledge, this is the first report of odc overexpression in D. innoxia leading to higher scopolamine yield.     

Photosynthetic and transpiration responses of <Emphasis Type="Italic">in vitro</Emphasis>-regenerated <Emphasis Type="Italic">Solanum nigrum</Emphasis> L. plants to <Emphasis Type="Italic">ex vitro</Emphasis> adaptation

In vitro regeneration of black nightshade (Solanum nigrum L.) plants was achieved through callus-mediated shoot organogenesis followed by 30 d indoor ex vitro adaptation to nutritional stress under environmental ambience and thereafter 6-d outdoor acclimatization in pots prior to field establishment. Relevant physiological parameters including pigment content, chlorophyll a fluorescence, net photosynthetic rate (P _N), transpiration rate (E), and stomatal conductance (g _s) of in vitro-regenerated plants were investigated during the course of ex vitro adaptation. During the first 4 d of indoor transplantation to potting substrate, there was a marginal reduction in the leaf chlorophyll and carotenoid contents but P _N and E were strongly reduced. The stomatal conductance and E/P _N ratio were significantly higher in plants up to 20 d of indoor adaptation than those of comparable age grown naturally from seeds. The shape of the OJIP fluorescence transient varied significantly with acclimatization, and the maximum change was observed at 2.0 ms. The 2.0 ms variable fluorescence (V _j), 30 ms relative fluorescence (M ₀), photon trapping probability (TR₀/Abs), and photosystem II (PSII) trapping rate (TR₀/RC) showed initial disturbance and subsequent stabilization during 30 d of indoor acclimatization. Energy dissipation (DI₀/RC) and electron transport probability (ET₀/TR₀) showed an initial phase of increase during the 4 d after plants were transplanted outdoors. During the 6-d outdoor acclimatization after transfer of plants to soil, no significant change in total chlorophylls and carotenoids, E, and g _s were observed, but P _N improved after reduction on the first d. The OJIP-derived parameters experienced change on the first d but were stabilized quickly thereafter. There was no significant difference between outdoor acclimatized plants and those of the seed-grown plants of comparable age with respect to photosynthetic and fluorescence parameters. Direct transfer of plants without indoor acclimatization, however, showed a completely different trend with respect to P _N, E, and OJIP fluorescence transients. The bearing of this study on optimizing micropropagation is discussed.     

Mycotoxin production by different ochratoxigenic <Emphasis Type="Italic">Aspergillus</Emphasis> and <Emphasis Type="Italic">Penicillium</Emphasis> species on coffee- and wheat-based media

Ochratoxin A (OTA) is one of the most widespread mycotoxins, and is produced by several Aspergillus or Penicillium species. Human exposure to OTA is mainly by intake of contaminated food, with cereal products, followed by coffee and red wine as the main sources of OTA. In this study, the OTA production of four ochratoxigenic fungi (two Aspergillus and two Penicillium species) was investigated in four different media, i.e. wheat and coffee model media as food-based media and two standard laboratory media (malt extract glucose agar, MEA and yeast extract sucrose agar, YES). Colony growth was documented and OTA concentrations in cultures were determined at day 2, 4 and 8 of incubation at 25°C by high-performance thin-layer chromatography (HPTLC) and high-performance liquid chromatography (HPLC). OTA production clearly depended upon time of incubation, fungal species, and medium composition. On coffee based medium, moderate OTA levels were produced by A. ochraceus BFE635 (9.8 μg/g) and by A. niger BFE632 (10.6 μg/g) on day 8 of incubation. In wheat-based medium, these strains produced much more OTA than in coffee. The highest OTA concentration (83.8 μg/g on day 8) was formed by A. ochraceus BFE635 followed by the other Aspergillus niger BFE632 (49 μg/g). Lower OTA levels were produced by P. verrucosum BFE550 and P. nordicum BFE487, in both wheat and in YES medium, whilst OTA was hardly detectable in coffee and in MEA in case of P. nordicum. Colony growth of the tested strains on different media was not indicative of OTA production. Guttation droplets developed on wheat-based medium with the Aspergillus strains within a week, and this phenomenon coincided with the high OTA amounts formed by these species. Results from this study add to our knowledge on the behaviour of ochratoxigenic fungal species when cultured on food based media.     

Studies on nutritional requirement for the culture of lichen <Emphasis Type="Italic">Ramalina nervulosa</Emphasis> and <Emphasis Type="Italic">Ramalina pacifica</Emphasis> to enhance the production of antioxidant metabolites

In the present report, nutritional requirement for the culture of two lichen species Ramalina nervulosa and Ramalina pacifica were studied in order to enhance their growth rate and antioxidant metabolite production. Extract of R. nervulosa cultured in Bold’s basal medium (BBM) showed higher antioxidant activity than R. pacifica cultured in Murashige and Skoog (MS) medium. The lichen species were sub-cultured in standardized nutrient media. R. nervulosa in BBM (1% glucose, 50 ppb asparagines, pH 6.5) yielded 2.76 g biomass with 26.18 mg sekikaic acid, 24.32 mg usnic acid/g dry biomass in a period of 60 days. R. pacifica in MS media (3% sucrose, 100 ppb thiamine, pH 5.9) yielded 3.54 g biomass and 58.92 mg salazinic acid, 40.16 mg usnic acid in the same time period. The standardized culture conditions implemented on bioreactor, R. nervulosa yielded 17.7 g biomass with the production of sekikaic acid 122.8 mg, usnic acid 75.4 mg in 4.5 days. R. pacifica produced 10.3 g biomass along with salazinic acid 200 mg and usnic acid 136.8 mg in the same duration. Lichen secondary metabolites produced in bioreactor showed moderate antioxidant activity; sekikaic acid 42% to 56.4%; salazinic acid 33.6% to 41.9% and usnic acid 19.9% to 29.5%.     

Removal of hexavalent chromium by fungal biomass of <Emphasis Type="Italic">Mucor racemosus</Emphasis>: influencing factors and removal mechanism

This study reported the hexavalent chromium removal by untreated Mucor racemosus biomass and the possible mechanism of Cr (VI) removal to the biomass. The optimum pH, biomass dose, initial Cr (VI) concentration and contact time were investigated thoroughly to optimize the removal condition. The metal removal by the biomass was strongly affected by pH and the optimum pH ranged from 0.5 to 1.0. The residual total Cr was determined. It was found that dichromate reduction occurred at a low very low pH value. At biomass dose 6 g/l, almost all the Cr (VI) ions were removed in the optimum condition. Higher removal percentage was observed at lower initial concentrations of Cr (VI) ions, while the removal capacity of the biomass linearly depended on the initial Cr (VI) concentration. More than half of Cr (VI) ions were diminished within 1 h of contact and removal process reached a relative equilibrium in approximately 8 h. Almost all of the Cr (VI) ions were removed in 24 h when initial concentrations were below 100 mg/l. The equilibrium data were fitted in to the Langmuir and the Freundlich isotherm models and the correlated coefficients were gained from the models. A Fourier transform infrared spectra was employed to elucidate clearly the possible biosorption mechanism as well.     

Lead Affects Apoptosis and Related Gene <Emphasis Type="Italic">XIAP</Emphasis> and <Emphasis Type="Italic">Smac</Emphasis> Expression in the Hippocampus of Developing Rats

Lead (Pb) exposure poses devastating effects on central nervous system development of children. To replicate aspects of this neurotoxicity, we examined the effect of lead on the expression of apoptosis and of apoptosis-related genes, XIAP (X chromosome-linked inhibitor of apoptosis protein) and Smac (second mitochondrial activator of caspase), in the hippocampus of developing rats. A total of 48 rats (30-day old) were randomly divided into four groups for intragastrical perfusion of lead acetate [Pb(Ac)₂]: untreated, low (2 mg/kg/d), medium (20 mg/kg/d), and high (200 mg/kg/d) dose groups. Pb content was determined in blood, and the apoptosis indexes and XIAP and Smac gene expression were analyzed in the hippocampus. There was a significant difference in apoptosis indexes (AI) between the exposed and control groups (p < 0.01). AI was highest in the high exposure group. XIAP gene expression was reduced in the exposed groups and the expression was negatively correlated with blood lead levels (BLLs) (p < 0.05). But the four groups did not differ in the expression of Smac (p > 0.05). Our data indicate that exposure to Pb(Ac)₂ caused a dose-dependent and significant increase of apoptosis in the hippocampus of developing rats through depressing the expression of the XIAP but not the Smac genes.     

Growth and carrageenan quality of <Emphasis Type="Italic">Kappaphycus striatum</Emphasis> var. <Emphasis Type="Italic">sacol</Emphasis> grown at different stocking densities,duration of culture and depth

Kappaphycus striatum var. sacol was grown in two separate studies: (1) at two stocking densities, and (2) at four different depths, each for three different durations of culture (30, 45 and 60 days) in order to determine the growth rate of the seaweed and evaluate the carrageenan content and its molecular weight. The results demonstrated that stocking density, duration of culture and depth significantly (P < 0.01) affected the growth rate, carrageenan content and molecular weight of K. striatum var. sacol. Decreasing growth rate was observed at both stocking densities and at four depths as duration of culture increased. A lower stocking density (500 g m⁻¹line⁻¹) showed a higher growth rate for the shortest durations, i.e. 30 days, as compared to those grown at a higher density. Likewise, decreasing growth rate was observed as depth increased, except at 50 cm after 60 days of culture. A 45-day culture period produced the highest molecular weight at both stocking densities (500 g m⁻¹line⁻¹ = 1,079.5 ± 31.8 kDa, 1,000 g m⁻¹line⁻¹ = 1,167 ± 270.6 kDa). ‘Sacol’ grown for 30 days at 50 cm (1,178 kDa) to 100 cm (1,200 kDa) depth showed the highest values of molecular weight of carrageenan extracted. The results suggested that K. striatum var. sacol is best grown at a stocking density of 500 g m⁻¹line⁻¹, at a depth of 50–100 cm, and for a duration of 30 days in order to provide the highest growth rate, carrageenan content and molecular weight.     

<Emphasis Type="Italic">Marchantia polymorpha</Emphasis> L.: A Bioaccumulator

Mussoorie in the western Himalayas of India, a popular tourist destination, was selected for air pollution monitoring. The study was carried out applying thalloid liverwort species Marchantia polymorpha L. as a tool. Moss bags containing M. polymorpha was transplanted at residential, highly and less polluted areas to obtain comprehensive and comparative data. Lead content, along with some essential micronutrients viz. zinc, manganese, and copper, as well as some physiological parameters total chlorophyll, sugar, protein, catalase and peroxidase activity were analyzed. The highest accumulation of lead (Pb) in M. polymorpha plants was highest in summer season , i.e., 2276 μg/g dry weight. The correlation of metals and physiological parameters has been made to get clear view of the effects of lead on physiology and essential micronutrients.     

Nickel(II) biosorption by <Emphasis Type="Italic">Rhodotorula glutinis</Emphasis>

The present study reports the feasibility of using Rhodotorula glutinis biomass as an alternative low-cost biosorbent to remove Ni(II) ions from aqueous solutions. Acetone-pretreated R. glutinis cells showed higher Ni(II) biosorption capacity than untreated cells at pH values ranging from 3 to 7.5, with an optimum pH of 7.5. The effects of other relevant environmental parameters, such as initial Ni(II) concentration, shaking contact time and temperature, on Ni(II) biosorption onto acetone-pretreated R. glutinis were evaluated. Significant enhancement of Ni(II) biosorption capacity was observed by increasing initial metal concentration and temperature. Kinetic studies showed that the kinetic data were best described by a pseudo-second-order kinetic model. Among the two-, three-, and four-parameter isotherm models tested, the Fritz-Schluender model exhibited the best fit to experimental data. Thermodynamic parameters (activation energy, and changes in activation enthalpy, activation entropy, and free energy of activation) revealed that the biosorption of Ni(II) ions onto acetone-pretreated R. glutinis biomass is an endothermic and non-spontaneous process, involving chemical sorption with weak interactions between the biosorbent and Ni(II) ions. The high sorption capacity (44.45 mg g⁻¹ at 25°C, and 63.53 mg g⁻¹ at 70°C) exhibited by acetone-pretreated R. glutinis biomass places this biosorbent among the best adsorbents currently available for removal of Ni(II) ions from aqueous effluents.     

Introduction of <Emphasis Type="Italic">OsglyII</Emphasis> gene into <Emphasis Type="Italic">Oryza sativa</Emphasis> for increasing salinity tolerance

Mature seed-derived embryogenic calli of indica rice (Oryza sativa L. cv. PAU201) were induced on semisolid Murashige and Skoog medium supplemented with 2.5 mg dm⁻³ 2,4-dichlorophenoxyacetic acid + 0.5 mg dm⁻³ kinetin + 560 mg dm⁻³ proline + 30 g dm⁻³ sucrose + 8 g dm⁻³ agar. Using OsglyII gene, out of 3180 calli bombarded, 32 plants were regenerated on medium containing hygromycin (30 mg dm⁻³). Histochemical GUS assay of the hygromycin selected calli revealed GUS expression in 50 % calli. Among the regenerants, 46.87 % were GUS positive. PCR analysis confirmed the presence of the transgene of 1 kb in 60 % of independent plants. Further, these plants have been grown to maturity in glasshouse. In vitro screening for salt tolerance showed increase in fresh mass of OsglyII putative transgenic calli (185.4 mg) as compared to control calli (84.2 mg) on 90 mM NaCl after 15 d. When exposed to 150 mM NaCl, OsglyII putative transgenic plantlets showed normal growth while the non-transgenic control plantlets turned yellow and finally did not survive.     

Enhancement of artemisinin content and biomass in <Emphasis Type="Italic">Artemisia annua</Emphasis> by exogenous GA<Subscript>3</Subscript> treatment

Artemisinin is a promising and potent antimalarial drug naturally produced by the plant Artemisia annua L. but in very low yield. Its artemisinin content is known to be greatly affected by both genotype and environmental factors. In this study, the production of artemisinin and leaf biomass in Artemisia annua L. was significantly increased by exogenous GA₃ treatment. The effect of GA₃ application on expression of proposed key enzymes involved in artemisinin yield was examined in both wild type (007) and FPS-overexpression (253-2) lines of A. annua. In the wild type (007) at 6 h post GA₃ application there was an abrupt rise in FPS, ADS and CYP71AV1 expression and at 24 h a temporary and significant peak in artemisinin (1.45-fold higher than the control). After GA₃ application in line 253-2, there was a dramatic rise in expression of FPS at 3 h, CYP71AV1 at 9 h and ADS at 72 h and accumulation of artemisinin after 7 days, which was a delay when compared with the wild type plant. Thus, increased artemisinin content from exogenous GA₃ treatment was associated with increased expression of key enzymes in the artemisinin biosynthesis pathway. Interestingly, exogenous GA₃ continuously enhanced artemisinin content from the vegetative stage to flower initiation in both plant lines and gave significantly higher leaf biomass than in control plants. Consequently, the artemisinin yield in GA₃-treated plants was much higher than in control plants. Although the maximum artemisinin content was found at the full blooming stage [2.1% dry weight (DW) in 007 and 2.4% DW in 253-2], the highest artemisinin yield in GA₃-treated plants was obtained during the flower initiation stage (2.4 mg/plant in 007 and 2.3 mg/plant in 235-2). This was 26.3 and 27.8% higher, respectively, than in non-treated plants 007 and 253-2. This study showed that exogenous GA₃ treatment enhanced artemisinin production in pot experiments and should be suitable for field application.     

Fed-batch production of a bioflocculant from <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

The constant-rate fed-batch production of the polygalacturonic acid bioflocculant REA-11 was studied. A controlled sucrose-feeding strategy resulted in a slight improvement in biomass and a 7% reduction in flocculating activity compared with the batch process. When fed with a 3 g l⁻¹ urea solution, the flocculating activity was enhanced to 720 U ml⁻¹ in 36 h. High cell density (2.12 g l⁻¹) and flocculating activity (820 U ml⁻¹) were obtained in a 10-l fermentor by feeding with a sucrose-urea solution, with values of nearly two times and 50% higher than those of the batch process, respectively. Moreover, the residual sucrose declined to 2.4 g l⁻¹, and residual urea decreased to 0.03 g l⁻¹. Even higher flocculating activity of 920 U ml⁻¹ and biomass of 3.26 g l⁻¹ were obtained by feeding with a sucrose-urea solution in a pilot scale fermentation process, indicating the potential industrial utility of this constant-rate feeding strategy in bioflocculant production by Corynebacterium glutamicum.     

Biomass production of<Emphasis Type="Italic">Anoectochilus formosanus</Emphasis> hayata in a bioreactor system

We investigated the factors that affect biomass production fromAnoectochilus formosanus in a bioreactor system. Those factors included inoculum size, initial sucrose concentration, media supplements, photosynthetic photon flux density (PPFD), and cuIturing methods. An inoculum size of 8 g L⁻¹ was most suitable for shoot proliferation; biomass accumulation was optimized when the medium was supplemented with 3% sucrose compared with sucrose-free media or those containing concentrations of 6% or 9%. This accumulation also was enhanced under a PPFD of 50 μmol m² s⁻¹. Likewise, the addition of coconut water (50 mL L⁻¹) plus activated charcoal (0.5 mg L⁻¹) to our Hyponex medium proved most beneficial. Comparative studies among three bioreactor systems — continuous immersion, raft (net), and temporary immersion (the ebb and flood system) — revealed that shoot proliferation and biomass accumulation were more efficient when culturing was performed under continuous immersion.     

Micropropagation of the Thai orchid <Emphasis Type="Italic">Grammatophyllum speciosum</Emphasis> blume

Protocorm-like bodies (PLBs) were induced from shoot tips of Grammatophyllum speciosum, a Thai orchid. The highest frequency of PLBs (93%) were observed on explants incubated on 1/2-Murashige and Skoog (MS) liquid medium containing 2% (w/v) sucrose without any plant growth regulators (PGRs). Tests with different carbon sources compared to sucrose revealed that maltose promoted the highest relative growth of G. speciosum PLBs (7-fold increase), while trehalose and sucrose yielded 5-fold and 4-fold increases, respectively. In 1/2 MS liquid medium, addition of 15 mg/l of chitosan promoted a 7-fold increase in PLB growth while 25 mg/l promoted a 4-fold increase. However, the relative growth rate in solid culture was significantly lower than that in liquid culture. In addition, chitosan supplementation in solid medium promoted shoot formation but not rooting. Plantlet regeneration was induced using a combination of NAA and BA supplementation in 1/2 MS solid medium with optimum induction shoot and root formation at 2.0 mg/l NAA and 1.0 mg/l BA. Using this protocol, approximately 8 months was required to obtain a hundred plantlets from one shoot tip. The plantlets showed no changes in ploidy when tested by flow cytometry.