A comparison of the toxic and sub-lethal effects of fluvalinate and esfenvalerate on the twospotted spider mite (Acari: Tetranychidae)

There was no difference in the direct toxicity of fluvalinate and esfenvalerate to twospotted spider mite (TSSM), Tetranychus urticae Koch. adults. The residual toxicity LC₅₀ of esfenvalerate was lower. Neither pyrethroid was toxic (<10% mortality) to TSSM eggs or adults at their recommended field concentrations. Fluvalinate was twice as toxic (45% mortality) than esfenvalerate to TSSM larvae at 0.01 g.a.i L^-1. The toxicity of the pyrethroids to TSSM protonymphs and deutonymphs was similar (16–28% mortality at 0.1 g a.i. L^-1). Dispersal from the treated surface was the main response to both pyrethroids by TSSM protonymphs, deutonymphs and adults. Maximum run-off by TSSM adults from fluvalinate and esfenvalerate treated surfaces was found with 0.01 and 0.005 g a.i. L^-1 respectively. Spin-down from pyrethroid treated surfaces was positively correlated with concentration. Oviposition was negatively correlated with concentration. Fluvalinate caused greater reductions in oviposition than esfenvalerate. Both pyrethroids reduced TSSM development rate from larval, protonymph and deutonymph stages, but fluvalinate caused larger reductions. Both pyrethroids prevented mating: for ten days oviposition 93% and 98% of offspring were male for esfenvalerate and fluvalinate respectively at 0.1 g a.i. L^-1. These findings are discussed with respect to the incidence of pyrethroid induced mite outbreaks.     

Improving intracellular production of recombinant protein in <Emphasis Type="Italic">Pichia pastoris</Emphasis> using an optimized preinduction glycerol-feeding scheme

High-cell-density production of recombinant growth hormone of Lateolabrax japonicus (rljGH) expressed intracellularly in Pichia pastoris was investigated. In the regular strategy of induction at a cell density of 160 g l⁻¹, short duration of intracellular rljGH accumulation (17 h) resulted in a low final cell density of 226 g l⁻¹. Thus, a novel strategy of induction at a cell density of 320 g l⁻¹ was investigated. In this strategy, the preinduction glycerol-feeding scheme had a significant effect on the post-induction production. Constant glycerol feeding led to a decrease of the specific rljGH production and specific production rate because of low preinduction specific growth rate. This decrease was avoided by exponential glycerol feeding to maintain a preinduction specific growth rate of 0.16 h⁻¹. The results from exponential glycerol feeding indicated that the rljGH production depended on the preinduction specific growth rate. Moreover, mixed feeding of methanol and glycerol during induction improved the specific production rate to 0.07 mg g⁻¹ h⁻¹ from 0.043 mg g⁻¹ h⁻¹. Consequently, both high cell density (428 g l⁻¹) and high rljGH production could be achieved by the novel strategy: growing the cells at the specific growth rate of 0.16 h⁻¹ to the cell density of 320 g l⁻¹ and inducing the expression by mixed feeding.     

Comparative toxic and sublethal effects of fluvalinate on two-spotted spider mite and European red mite

Four fluvalinate formulations differed in their residual toxicity to female two-spotted spider mite (TSM), Tetranychus urticae adults; the emulsifiable concentrate (EC) was the most toxic. In contrast, there was little difference in toxicity between the formulations with the European red mite (ERM) Panonychus ulmi with the exception of the EC formulation which was the least toxic. Fluvalinate 2F caused minimal (<10%) TSM and ERM egg mortality. Fluvalinate 2F was more toxic and caused greater larval dispersal for the TSM compared to the ERM at the field concentration and below. The toxicity of fluvalinate 2F to TSM and ERM protonymphs, deutonymphs and adults was low, approximately <20% at field concentration. Dispersal was the main response to fluvalinate and this was positively correlated with increasing concentration. The combined mortality and dispersal LC₅₀ was five times lower for ERM protonymphs and adults, but 11 times higher for ERM deutonymphs compared to equivalent TSM life stages. Fluvalinate 2F reduced TSM development from the protonymph and deutonymph stages to a greater extent compared to the ERM. The mortality response to fluvalinate 2F was unaffected by host type (peach or apple) for the TSM whereas ERM mortality was higher on apple compared to peach. TSM dispersal was higher from apple compared to peach whereas ERM dispersal was similar on both host types. Oviposition by both mite species was lower on apple than peach leaves. A 1 h exposure to fluvalinate 2F reduced ERM oviposition for 12 days.     

Characterization of a thermophilic sulfur oxidizing enrichment culture dominated by a Sulfolobus sp. obtained from an underground hot spring for use in extreme bioleaching conditions

A thermoacidophilic elemental sulfur and chalcopyrite oxidizing enrichment culture VS2 was obtained from hot spring run-off sediments of an underground mine. It contained only archaeal species, namely a Sulfolobus metallicus-related organism (96% similarity in partial 16S rRNA gene) and Thermoplasma acidophilum (98% similarity in partial 16S rRNA gene). The VS2 culture grew in a temperature range of 35–76°C. Sulfur oxidation by VS2 was optimal at 70°C, with the highest oxidation rate being 99 mg S⁰ l⁻¹ day⁻¹. At 50°C, the highest sulfur oxidation rate was 89 mg l⁻¹ day⁻¹ (in the presence of 5 g Cl⁻ l⁻¹). Sulfur oxidation was not significantly affected by 0.02–0.1 g l⁻¹ yeast extract or saline water (total salinity of 0.6 M) that simulated mine water at field application sites with availability of only saline water. Chloride ions at a concentration above 10 g l⁻¹ inhibited sulfur oxidation. Both granular and powdered forms of sulfur were bioavailable, but the oxidation rate of granular sulfur was less than 50% of the powdered form. Chalcopyrite concentrate oxidation (1% w/v) by the VS2 resulted in a 90% Cu yield in 30 days.     

Effects of water level fluctuation on the growth ofNelumbo nucifera Gaertn. in Lake Kasumigaura,Japan

Investigations were made of the growth ofNelumbo nucifera, an aquatic higher plant, in a natural stand in Lake Kasumigaura. A rise of 1.0 m in the water level after a typhoon in August 1986 caused a subsequent decrease in biomass ofN. nucifera from the maximum of 291 g d.w. m⁻² in July to a minimum of 75 g d.w. m⁻². The biomass recovered thereafter in shallower regions. The underground biomass in October tended to increase toward the shore. The total leaf area index (LAI) is the sum of LAI of floating leaves and emergent leaves. The maximum total LAI was 1.3 and 2.8 m² m⁻² in 1986 and 1987, respectively. LAI of floating leaves did not exceed 1 m² m⁻². The elongation rates of the petiole of floating and emergent leaves just after unrolling were 2.6 and 3.4 cm day⁻¹, respectively. The sudden rise in water level (25 cm day⁻¹) after the typhoon in August 1986 caused drowning and subsequent decomposition of the mature leaves. Only the young leaves were able to elongate, allowing their laminae to reach the water surface. The fluctuation in water level, characterized by the amplitude and duration of flooding and the time of flooding in the life cycle, is an important factor determining the growth and survival ofN. nucifera in Lake Kasumigaura.     

Two-stage glycerol feeding for enhancement of recombinant hG-CSF production in a fed-batch culture of Pichia pastoris

Recombinant hG-CSF was expressed in Pichia pastoris under the control of the AOX1 promoter. In this study, the glycerol feeding rate was adjusted to achieve the maximum attainable specific growth rate before induction. Using a two-stage glycerol feeding method, the specific growth rate was changed from a maximum value of 0.21 h⁻¹ (at the beginning of feeding) to 0.15 h⁻¹ prior to induction. With this approach, the final dry cell wt and rhG-CSF yield achieved was close to 120 g l⁻¹ and 320 mg l⁻¹, respectively. Our study found that the two-stage feeding method allowed the overall productivity of rhG-CSF to increase 2.9 times that of the conventional fed-batch method.     

Photoinhibition-induced reduction in photosynthesis is alleviated by abscisic acid,cytokinin and brassinosteroid in detached tomato leaves

Detached leaves of tomato (Lycopersicon esculentum Mill.) experienced photoinhibition associated with sharp reductions in net photosynthetic rate (Pn), quantum efficiency of PSII (Φ_PSII) and photochemical quenching (qP) even though they were exposed to mild light intensity (400 μmol m⁻² s⁻¹ PPFD) at 28°C. Photoinhibition and the reduction in Pn, Φ_PSII and qP, however, were significantly alleviated by 1 mg l⁻¹ ABA, 0.1 mg l⁻¹ N-(2-chloro-4-pyridyl)-N′-phenylurea (CPPU) and 0.01 mg l⁻¹ 24-epibrassinolide (EBR). Higher concentrations, however, reduced the effects or even exacerbated the occurrence of photoinhibition. Superoxide dismutase and ascorbate peroxidase activity in leaves increased with the increases in ABA concentration within 1–100 mg l⁻¹, CPPU concentration within 0.1–10 mg l⁻¹ and EBR concentration within 0.01–1.0 mg l⁻¹. Catalase and guaiacol peroxidase activity also increased with the increase in EBR concentration but CPPU and ABA treatments at higher concentrations caused a decrease. Malondialdehyde (MDA) content decreased with the increase in CPPU concentration. ABA and EBR, however, decreased MDA concentration only at 1 and 0.01 mg l⁻¹, respectively. In conclusion, detached leaves had increased sensitivity to PSII photoinhibition. Photoinhibition-induced decrease in photosynthesis, however, was significantly alleviated by EBR, CPPU and ABA at a proper concentration.     

Effect of jasmonic acid on in vitro explant growth and microtuberization in potato

The shoot fresh mass, root length and root numbers of two potato (Solanum tuberosum L.) cultivars Favorita and Helanwuhua were increased significantly by the application of 0.2 – 2 mg dm⁻³ jasmonic acid (JA) in the Murashige and Skoog medium. However, the growth of potato explants was inhibited by JA at high concentrations (20 – 50 mg dm⁻³). Chlorophyll content in explant leaves decreased with an increase in the concentration of JA. In leaves treated with 0.2 mg dm⁻³ JA acid peroxidase activity increased, while in the leaves treated with more than 2 mg dm⁻³ JA peroxidase activity decreased. Under the dark, the microtuber numbers, fresh mass and percentage of big microtubers of two potato cultivars were not promoted by the application of 0.2 – 50 mg dm⁻³ JA.     

Production of lipase by high cell density fed-batch culture of Candida cylindracea

Candida cylindracea NRRL Y-17506 was grown to produce extracellular lipase from oleic acid as a carbon source. Through flask cultures, it was found that the optimum initial oleic acid concentration for cell growth was 20 g l⁻¹. However, high initial concentrations of oleic acid up to 50 g l⁻¹ were not inhibitory. The highest extracellular lipase activity obtained in flask culture was 3.0 U ml⁻¹ after 48 h with 5 g l⁻¹ of initial oleic acid concentration. Fed-batch cultures (intermittent and stepwise feeding) were carried out to improve cell concentration and lipase activity. For the intermittent feeding fed-batch culture, the final cell concentration was 52 g l⁻¹ and the extracellular lipase activity was 6.3 U ml⁻¹ at 138.5 h. Stepwise feeding fed-batch cultures were carried out to simulate an exponential feeding and to investigate the effects of specific growth rate (0.02, 0.04 and 0.08 h⁻¹) on cell growth and lipase production. The highest final cell concentration obtained was 90 g l⁻¹ when the set point of specific growth rate (μ_set) was 0.02 h⁻¹. High specific growth rate (0.04 and 0.08 h⁻¹) decreased extracellular lipase production in the later part of fed-batch cultures due to build-up of the oleic acid oversupplied. The highest extracellular lipase activity was 23.7 U ml⁻¹ when μ_set was 0.02 h⁻¹, while the highest lipase productivity was 0.31 U ml⁻¹ h⁻¹ at μ_set of 0.08 h⁻¹.     

Magnesium deficiency of kiwifruit (Actinidia deliciosa)

Magnesium deficiency was associated with large yield reductions in a five-year-old commercial kiwifruit (Actinidia deliciosa) orchard. The effect on yield resulted primarily from a reduction in fruit numbers, there being no difference in mean fruit weight between fruit harvested from affected and unaffected vines. Magnesium deficiency had no deleterious effect on postharvest storage characteristics of fruit stored at 0.5–1°C for 18 weeks; fruit from deficient vines were firmer but had slightly lower soluble solids than fruit from control vines. Although deficiency symptoms were first observed on the basal leaves of the non-fruiting shoots mid season, indications of the impending deficiency could be established very early in the season using foliar analysis. Magnesium concentrations in youngest fully expanded leaves (YFEL) on the affected vines were less than 2.0 g kg⁻¹ DM four weeks after budbreak and remained below this value for the rest of the season; concentrations in YFEL on unaffected vines did not decrease below this value and gradually increased after fruitset to 4.5 g kg⁻¹ DM at harvest. To avert potential production losses, it is suggested that soluble magnesium fertilizers (containing at least 200 kg ha⁻¹ Mg) should be broadcast early in the season if foliar magnesium concentrations less than 2.0 gkg⁻¹ DM are measured four–six weeks after budbreak.     

Effects of Progesterone, β-Estradiol,and Androsterone on the Changes of Inorganic Element Content in Barley Leaves

The present study was performed to determine the changes in inorganic element content in barley leaves of mammalian sex hormones (MSH). Barley leaves were sprayed with 10⁻⁴, 10⁻⁶, 10⁻⁹, 10⁻¹², 10⁻¹⁵ M concentrations of progesterone, β-estradiol, and androsterone at 7th day after sowing. The plants were harvested at the end of 18 days after treatment with MSH solutions. The inorganic element concentrations were determined using wavelength dispersive X-ray fluorescence spectroscopy technique. Although the all MSH concentrations significantly (p < 0.05) increased the concentrations of calcium, magnesium, phosphorus, sulfur, copper, manganese, aluminum, zinc, iron, potassium, and chlorine, it decreased those of sodium concentration in barley leaves. The maximum changes in the element concentrations were obtained at 10⁻⁹ M for plant leaves treated with progesterone, 10⁻⁶ M for plant leaves treated with β-estradiol and androsterone. The present study elucidated that MSH significantly (p < 0.05) affected the inorganic element concentrations in barley leaves.     

Production of 1,3-Propanediol by Clostridium butyricum VPI 3266 in continuous cultures with high yield and productivity

The effects of dilution rate and substrate feed concentration on continuous glycerol fermentation by Clostridium butyricum VPI 3266, a natural 1,3-propanediol producer, were evaluated in this work. A high and constant 1,3-propanediol yield (around 0.65 mol/mol), close to the theoretical value, was obtained irrespective of substrate feed concentration or dilution rate. Improvement of 1,3-propanediol volumetric productivity was achieved by increasing the dilution rate, at a fixed feed substrate concentration of 30, 60 or 70 g l⁻¹. Higher 1,3-propanediol final concentrations and volumetric productivities were also obtained when glycerol feed concentration was increased from 30 to 60 g l⁻¹, at D=0.05–0.3 h⁻¹, and from 60–70 g l⁻¹, at D=0.05 and 0.1 h⁻¹·30 g l⁻¹ of 1,3-propanediol and the highest reported value of productivity, 10.3 g l⁻¹ h⁻¹, was achieved at D=0.30 h⁻¹ and 60 g l⁻¹ of feed glycerol. A switch to an acetate/butyrate ratio higher than one was observed for 60 g l⁻¹ of feed glycerol and a dilution rate higher than 0.10 h⁻¹; moreover, at D=0.30 h⁻¹ 3-hydroxypropionaldehyde accumulation was observed for the first time in the fermentation broth of C. butyricum.     

Energy flow and subsidies associated with the complex life cycle of ambystomatid salamanders in ponds and adjacent forest in southern Illinois

Breeding adults and metamorphosing larval amphibians transfer energy between freshwater and terrestrial ecosystems during seasonal migrations and emergences, although rarely has this been quantified. We intensively sampled ambystomatid salamander assemblages (Ambystoma opacum,A. maculatum, and A. tigrinum) in five forested ponds in southern Illinois to quantify energy flow associated with egg deposition, larval production, and emergence of metamorphosed larvae. Oviposition by female salamanders added 7.0–761.4 g ash-free dry mass (AFDM) year⁻¹ to ponds (up to 5.5 g AFDM m⁻² year⁻¹). Larval production ranged from 0.4 to 7.4 g AFDM m⁻² year⁻¹ among populations in three ponds that did not dry during larval development, with as much as 7.9 g AFDM m⁻² year⁻¹ produced by an entire assemblage. Mean larval biomass during cohort production intervals in these three ponds ranged from 0.1 to 2.3 g AFDM m⁻² and annual P/B (production/biomass) ranged from 4 to 21 for individual taxa. Emergent biomass averaged 10% (range=2–35%) of larval production; larval mortality within ponds accounted for the difference. Hydroperiod and intraguild predation limited larval production in some ponds, but emerging metamorphs exported an average of 70.0±33.9 g AFDM year⁻¹ (range=21.0–135.2 g AFDM year⁻¹) from ponds to surrounding forest. For the three ponds where larvae survived to metamorphosis, salamander assemblages provided an average net flux of 349.5±140.8 g AFDM year⁻¹ into pond habitats. Among all ponds, net flux into ponds was highest for the largest pond and decreased for smaller ponds with higher perimeter to surface area ratios (r ² =0.94, P<0.05, n=5). These results are important in understanding the multiple functional roles of salamanders and the impact of amphibian population declines on ecosystems. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Critical Levels of Selenium in Different Crops Grown in an Alkaline Silty Loam Soil Treated with Selenite-Se

Critical levels of selenium in raya (Brassica juncea Czern L.), maize (Zea mays L.), wheat (Triticum aestivum L.) and rice (Oryza sativa L.) were worked out by growing these crops in an alkaline silty loam soil treated with different levels of selenite-Se ranging from 1 to 25 μg g⁻¹ soil. Significant decrease in dry matter yield was observed above a level of 5 μg Se g⁻¹ soil in raya and maize; 4 μg Se g⁻¹ soil in wheat and 10 μg Se g⁻¹ soil in rice shoots. The critical level of Se in plants above which significant decrease in yield would occur was found to be 104.8 μg g⁻¹ in raya, 76.9 μg g⁻¹ in maize, 41.5 μg g⁻¹ in rice and 18.9 μg g⁻¹ in wheat shoots. Significant coefficients of correlation were observed between Se content above the critical level and dry matter yield of raya as well as rice (r = −0.99, P ≤ 0.01), wheat (r = −0.97, P ≤ 0.01) and maize ((r = −0.96, P ≤ 0.01). A synergistic relationship was observed between S and Se content of raya (r = 0.96, P ≤ 0.01), wheat (r = 0.89, P ≤ 0.01), rice (r = 0.85, P ≤ 0.01) and maize (r = 0.84, P ≤ 0.01). Raya, maize and rice absorbed Se in levels toxic for animal consumption (i.e. > 5 mg Se kg⁻¹) when the soil was treated with more than 1.5 μg Se g⁻¹. In case of wheat, application of Se more than 3 μg g⁻¹ soil resulted in production of toxic plants.     

Stem tissue phosphorus as an index of the phosphorus status of Banksia ericifolia L. f.

The effects of P fertilizer rate on shoot growth and the total P concentration of the whole shoot, new and mature leaves, symptom leaves and stems of Banksia ericifolia L. f., a P-sensitive species, were investigated in a six month greenhouse pot experiment. Shoot dry weight of plants growing in an Australian sedge peat, coarse sand and perlite potting mix (1:1:1) increased with up to 100 mg P L⁻¹ supplied as a six month controlled release P (0:18:0) fertilizer, but was reduced by toxicity at the highest application rate (200 mg P L⁻¹). Plants receiving this treatment developed chlorotic new and mature leaves. Leaf symptoms observed at rates of 60–100 mg P L⁻¹ were confined to old leaves and were related to the P concentration of the shoot. Growth was not affected at these rates. The P concentration of stems was strongly influenced by P supply. This tissue acted as a sink for excess P, helping to regulate the P concentration of leaves. The approximate range of P concentrations in stem tissue, associated with greater than 90% of maximum shoot dry weight, was 0.5–1.5 g P kg⁻¹ tissue dry weight. This was greater than that calculated for mature leaves (0.5–0.8 g kg⁻¹) or for whole shoots (0.5–1.2 g kg⁻¹). This wider range, and the capacity to store P in excess to requirement, makes the stem a better index tissue for plant P status than either leaves or whole shoots. This revised version was published online in June 2006 with corrections to the Cover Date.     

Ethanol production from sweet sorghum juice in batch and fed-batch fermentations by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Sweet sorghum juice supplemented with 0.5% ammonium sulphate was used as a substrate for ethanol production by Saccharomyces cerevisiae TISTR 5048. In batch fermentation, kinetic parameters for ethanol production depended on initial cell and sugar concentrations. The optimum initial cell and sugar concentrations in the batch fermentation were 1 × 10⁸ cells ml⁻¹ and 24 °Bx respectively. At these conditions, ethanol concentration produced (P), yield (Y _ps) and productivity (Q _p ) were 100 g l⁻¹, 0.42 g g⁻¹ and 1.67 g l⁻¹ h⁻¹ respectively. In fed-batch fermentation, the optimum substrate feeding strategy for ethanol production at the initial sugar concentration of 24 °Bx was one-time substrate feeding, where P, Y _ps and Q _p were 120 g l⁻¹, 0.48 g g⁻¹ and 1.11 g l⁻¹ h⁻¹ respectively. These findings suggest that fed-batch fermentation improves the efficiency of ethanol production in terms of ethanol concentration and product yield.     

Nitrate and ammonium accumulation in bermudagrass in relation to nitrogen fertilization and season

Seasonal changes in nitrate and ammonium concentrations were studied inCynodon dactylon (L.) Pers. plants grown for one year in the field in a Mediterranean area. Plants cultivated in a sandy loam soil were fertilized with nitrate-N or ammonium-N at two application rates (250 and 1000 kg N ha⁻¹ year⁻¹) and compared to controls with no added N. Plots were harvested every three weeks from May to November. Shoots were separated into leaves and stems and analyses carried out in both fractions. Nitrogen applications generally led to elevated nitrate concentrations both in leaves and stems at all sampling dates but had little influence on the ammonium concentrations of the tissues. Higher nitrate and ammonium concentrations were found in stems than in leaves, although no levels higher than 0.22% NO ₃ ⁻ −N and 0.10% NH ₄ ⁺ −N were detected in either fraction. Nitrate tended to accumulate mostly in autumn and spring whereas low accumulations were found in summer. Ammonium showed both in leaves and stems a progressive but limited accumulation throughout the period with a peak in October, followed by a strong decrease in November.     

Physiological responses induced by copper bioaccumulation in <Emphasis Type="Italic">Eichhornia crassipes</Emphasis> (Mart.)

Eichhornia crassipes (Mart.) has strong ability to remove Cu²⁺ from copper-contaminated water. Physiological responses in E. crassipes exposed to known concentrations of Cu²⁺ were examined in this study, and demonstrated that E. crassipes could accumulate 314 mg kg⁻¹ dry weight of Cu when exposed to 5 mg l⁻¹ of Cu²⁺ for periods up to 14 d. However, there were marked changes in physiology of the plant commencing at Cu²⁺ concentrations of 1 mg l⁻¹. Results of this study showed that E. crassipes could tolerate moderate concentrations (i.e. 0.5 mg l⁻¹) of Cu²⁺, without significant changes in photosynthetic pigment concentrations, while high concentrations (i.e. 5 and 10 mg l⁻¹) of Cu²⁺ resulted in substantial loss in pigment concentrations. Increases in malondiadehyde (MDA) content were also demonstrated in plant exposure to high Cu²⁺ concentrations. Soluble protein content increased to a level slightly higher than the control at <0.5 mg l⁻¹ of Cu²⁺, but then decreased with exposure to >1 mg l⁻¹ of Cu²⁺. Our results suggest that E. crassipes has a substantial capacity to accumulate copper when cultivated at moderate concentrations of Cu²⁺, without marked changes in its physiology. The findings indicate that E. crassipes is a promising possibility for phytoremediation of moderately Cu-contaminated water bodies. Handling editor: S. M. Thomaz     

Effect of irradiation intensity on cell growth and kalata B1 accumulation in <Emphasis Type="Italic">Oldenlandia affinis</Emphasis> cultures

Light irradiation had remarkable effects on callus growth of Oldenlandia affinis with an optimum intensity of 35 μmol m⁻² s⁻¹. Biosynthesis of kalata B1, the main cyclic peptide in O. affinis, was induced and triggered with rising irradiation intensities. The highest concentration of kalata B1, 0.49 mg g⁻¹ DW characterised by the maximum productivity of 3.88 μg per litre and day was analysed at 120 μmol m⁻² s⁻¹, although callus growth was repressed. The light saturation point was established to be 35 μmol m⁻² s⁻¹, where kalata B1 productivity was in a similar order (3.41 μg per day) due to the higher growth index. O. affinis suspension cultures were shown to accumulate comparable specific kalata B1 concentrations in a delayed growth associated production pattern. These were dependent on irradiation intensity (0.16 mg g⁻¹ at 2 μmol m⁻² s⁻¹; 0.28 mg g⁻¹ at 35 μmol m⁻² s⁻¹). The batch cultivation process resulted in a maximum productivity of 27.30 μg per litre and day with culture doubling times of 1.16 d⁻¹. Submers operation represented a 8-fold product enhancement compared to callus cultivation.     

Isolation of <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> mutants for heterologous protein production from a double proteinase gene disruptant

The recombinant Pichia pastoris harboring an improved methionine adenosyltransferase (MAT) shuffled gene was employed to biosynthesize S-adenosyl-l-methionine (SAM). Two l-methionine (l-Met) addition strategies were used to supply the precursor: the batch addition strategy (l-Met was added separately at three time points) and the continuous feeding strategies (l-Met was fed continuously at the rate of 0.1, 0.2, and 0.5 g l⁻¹ h⁻¹, respectively). SAM accumulation, l-Met conversion rate, and SAM productivity with the continuous feeding strategies were all improved over the batch addition strategy, which reached 8.46 ± 0.31 g l⁻¹, 41.7 ± 1.4%, and 0.18 ± 0.01 g l⁻¹ h⁻¹ with the best continuous feeding strategy (0.2 g l⁻¹ h⁻¹), respectively. The bottleneck for SAM production with the low l-Met feeding rate (0.1 g L⁻¹ h⁻¹) was the insufficient l-Met supply. The analysis of the key enzyme activities indicated that the tricarboxylic acid cycle and glycolytic pathway were reduced with the increasing l-Met feeding rate, which decreased the adenosine triphosphate (ATP) synthesis. The MAT activity also decreased as the l-Met feeding rate rose. The reduced ATP synthesis and MAT activity were probably the reason for the low SAM accumulation when the l-Met feeding rate reached 0.5 g l⁻¹ h⁻¹.