Dialysis cultures with immobilized hybridoma cells for effective production of monoclonal antibodies

An industrial scale reactor concept for continuous cultivation of immobilized animal cells (e.g. hybridoma cells) in a radial-flow fixed bed is presented, where low molecular weight metabolites are removed via dialysis membrane and high molecular products (e.g. monoclonal antibodies) are enriched. In a new nutrient-split feeding strategy concentrated medium is fed directly to the fixed bed unit, whereas a buffer solution is used as dialysis fluid. This feeding strategy was investigated in a laboratory scale reactor with hybridoma cells for production of monoclonal antibodies. A steady state monoclonal antibody concentration of 478 mg l^-1 was reached, appr. 15 times more compared to the concentration reached in chemostat cultures with suspended cells. Glucose and glutamine were used up to 98%. The experiments were described successfully with a kinetic model for immobilized growing cells. Conclusions were drawn for scale-up and design of the large scale system.Abbreviations: c_Glc – glucose concentration, mmol l^-1; c_Gln – glutamine concentration, mmol l^-1; c_Amm – ammonia concentration, mmol l^-1; c_Lac – lactate concentration, mmol l^-1; c_MAb – MAb concentration, mg l^-1; D – dilution rate, d^-1; D_i – dilution rate in the inner chamber of the membrane dialysis reactor, d^-1; D₀ – dilution rate in the outer chamber of the membrane dialysis reactor, d^-1; q*_FB,Glc – volume specific glucose uptake rate related to the fixed bed volume, mmol l_FB ^-1 h^-1; q*_FB,Gln – volume specific glutamine uptake rate related to the fixed bed volume, mmol l_FB ^-1 h^-1.     

Conversion of cellulose into fungal cell mass

Summary Chaetomium cellulolyticum (ATCC 32319) was cultivated on glucose, Avicel and/or Sigmacell in a 20-1 stirred tank batch reactor. The substrate (cellulose) concentration, the cell mass concentration (through protein and/or nitrogen content), reducing sugar concentration, the enzyme activity, the alkali consumption rate, the dissolved O₂ and CO₂ concentrations in the outlet gas were measured. The specific growth rate, the substrate yield coefficient, cell productivity, the oxygen consumption rate, the CO₂ production rate and the volumetric mass transfer coefficient were determined. At the beginning of the growth phase the oxygen utilization rate exhibits a sharp maximum. This maximum could be used to start process control. Because of the long lag phase periodic batch operation is recommended.Symbols CP cell protein concentration (g l^–1) - FPA FP enzyme activity (IU l^–1) - GP dissolved protein concentration (g l^–1) - IU international unit of enzyme activity - k_La volumetric mass tranfer coefficient (h^–1) - LG alkali (1 n NaOH) consumption (ml) - LGX specific alkali consumption rate per cell mass (ml g^–1 h^–1) - P cell mass productivity (g l^–1 h^–1) - specific oxygen consumption rate per cell mass (g g^–1 h^–1) - Q aeration rate (volumetric gas flow rate per volume of medium, vvm) (min^–1) - N impeller speed (revolution per minute, rpm) (min^–1) - S substrate concentration (g l^–1) - S⁰ S at t_F=0 (g l^–1) - S₀ S in feed (g l^–1) - SR acid consumption (ml) - TDW total dry weight (g l^–1) - T temperature (° C) - t_F cultivation time (h) - U substrate conversion - X cell mass concentration (g l^–1) - Y_X/S vield coefficient - specific growth rate (h^–1) - _m maximum specific growth rate (h^–1)     

Enzyme production in continuous cultivation by the thermophilic fungus, Thermomyces lanuginosus

The production of extracellular enzymes by the thermophilic fungus Thermomyces lanuginosus was studied in chemostat cultures at a dilution rate of 0.08 h^–1 in relation to variation in the ammonium concentration in the feed medium. Under steady state conditions, three growth regimes were recognised and the production of several extracellular enzymes from T. lanuginosus was recorded under different nutrient limitations ranging from nitrogen limitation to carbon/energy limitation. The range and the production of carbohydrate hydrolysing enzymes and lipase increased from Regime I (NH₄Cl 600 mg l^–1) to Regime III (NH₄CI 1200 mg l^–1), whereas production of protease was highest in Regime II (600 mg l^–1 < NH₄Cl <1200 mg l^–1).     

Improvement of the culture stability of non-anchorage-dependent animal cells grown in serum-free media through immobilization

A murine hybridoma cell line producing a monoclonal antibody against penicillin-G-amidase and a murine transfectoma cell line secreting a monovalent chimeric human/mouse Fab-antibody fragment were cultivated in three different media (serum-containing, low protein serum-free, and iron-rich protein-free) in flask cultures, stirred reactors and a fixed bed reactor. In static batch cultures in flasks both cell lines showed similar good growth in all three media.In suspension in a stirred reactor, the hybridoma cell line could be cultivated satisfactory only in serum-containing medium. In low protein serum-free medium, Pluronic F68 had to be added to protect the hybridoma cells against shear stress. But even with this supplement only batch, not chemostat mode was possible. In iron-rich protein-free medium the hybridoma cells grew also in continuous chemostat mode, but the stability of the culture was low. The transfectoma cell line did not grow in stirred reactors in any of the three media.Good results with both cell lines were obtained in fixed bed experiments, where the cells were immobilized in macroporous Siran^®-carriers. The media, which were optimized in flask cultures, could be used without any further adaptation in the fixed bed reactor. Immobilization improved the stability and reliability of cultures of non-adherent animal cells in serum-free media tremendously compared to suspension cultures in stirred reactors. The volume-specific glucose uptake rate, an, indicator of the activity of the immobilized cells, was similar in all three media. Deviations in the metabolism of immobilized and suspended cells seem to be mainly due to low oxygen concentrations within the macroporous carriers, where the cells are supplied with oxygen only by diffusion.List of symbols c substrate or product concentration mmol l^–1 - c₀ substrate or product concentration in the feed mmol l^–1 - c_Glc glucose concentration mmol l^–1 - c_Gln glutamine concentration mmol l^–1 - c_Amm ammonia concentration mmol l^–1 - c_Lac lactate concentration mmol l^–1 - c_FAB concentration of Fab# 10 antibody fragment g l^–1 - c_MAb monoclonal antibody concentration mg l^–1 - D dilution rate d^–1 - q cell-specific substrate uptake or metabolite production rate mmol cell^–1 h^–1 - q_Glc cell-specific glucose uptake rate mmol cell^–1 h^–1 - q_Gln cell-specific glutamine uptake rate mmol cell^–1 h^–1 - q_MAb cell-specific MAb production rate mg cell^–1 h^–1 - q^* volume-specific substrate uptake or metabolite production rate mmol l^–1 h^–1 - q^*FB volume-specific substrate uptake or metabolite production rate related to the fixed bed volume mmol l_FB ^–1 h^–1 - q^*FB,Glc volume-specific glucose uptake rate related to the fixed bed volume mmol l_FB ^–1 h^–1 - q^*FB,Gln volume-specific glutamine uptake rate related to the fixed volume mmol l_FB ^–1 h^–1 - q^*FB,MAb volume-specific MAb production rate related to the fixed volume mg l_FB ^–1 h^–1 - q^*FB,02 volume-specific oxygen uptake rate related to the fixed bed volume mmol l_FB ^–1 h^–1 - t time h - U superficial flow velocity mm s^–1 - V medium volume in the conditioning vessel of the fixed bed reactor l - V_FB volume of the fixed bed l - x_v viable cell concentration cells ml^–1 - y_Amm,Gln yield of Ammonia from glutamine - y_Lac,Glc yield of lactate from glucose - specific growth rate h^–1 - _d specific death rate h^–1     

Control of carbon mineralization to CH4 and CO2 in anaerobic,Sphagnum-derived peat from Big Run Bog,West Virginia

The mineralization of organic carbon to CH₄ and CO₂ inSphagnum-derived peat from Big Run Bog, West Virginia, was measured at 4 times in the year (February, May, September, and November) using anaerobic, peat-slurry incubations. Rates of both CH₄ production and CO₂ production changed seasonally in surface peat (0–25 cm depth), but were the same on each collection date in deep peat (30–45 cm depth). Methane production in surface peat ranged from 0.2 to 18.8 mol mol(C)^–1 hr^–1 (or 0.07 to 10.4 g(CH₄) g^–1 hr^–1) between the February and September collections, respectively, and was approximately 1 mol mol(C)^–1 hr^–1 in deep peat. Carbon dioxide production in surface peat ranged from 3.2 to 20 mol mol(C)^–1 hr^–1 (or 4.8 to 30.3 g(CO₂) g^–1 hr^–1) between the February and September collections, respectively, and was about 4 mol mol(C)^–1 hr^–1 in deep peat. In surface peat, temperature the master variable controlling the seasonal pattern in CO₂ production, but the rate of CH₄ production still had the lowest values in the February collection even when the peat was incubated at 19°C. The addition of glucose, acetate, and H₂ to the peat-slurry did not stimulate CH₄ production in surface peat, indicating that CH₄ production in the winter was limited by factors other than glucose degradation products. The low rate of carbon mineralization in deep peat was due, in part, to poor chemical quality of the peat, because adding glucose and hydrogen directly stimulated CH₄ production, and CO₂ production to a lesser extent. Acetate was utilized in the peat by methanogens, but became a toxin at low pH values. The addition of SO₄ ^2– to the peat-slurry inhibited CH4 production in surface peat, as expected, but surprisingly increased carbon mineralization through CH4 production in deep peat. Carbon mineralization under anaerobic conditions is of sufficient magnitude to have a major influence on peat accumulation and helps to explain the thin (< 2 m deep), old (> 13,000 yr) peat deposit found in Big Run Bog.     

Limnological control of brine shrimp population dynamics and cyst production in the Great Salt Lake, Utah

In the Great Salt Lake of Utah, the brine shrimp Artemia franciscanaKellogg is an important food resource for birds and they produce dormant cysts that are harvested and used extensively in the aquaculture industry. We analyzed the limnological factors controlling Artemia growth and cyst production over 12 months in 1994 and 1995. Laboratory experiments showed that inter-brood intervals were highly dependent on temperature and slightly on food level. At optimal temperatures and nutritious food, juveniles reached reproductive size within 7 d in the laboratory. In winter when temperatures were less than 3 °C, Artemia were absent from the lake, phytoplankton abundance was high (13 Chl a g l^–1), and the dominant grazers were ciliated protozoans. In the spring, cysts hatched when phytoplankton was abundant (15–30 g Chl a l^–1), and the Artemia grew and produced large clutches of ovoviviparous eggs. Estimated naupliar production from these eggs was 80 l^–1 from April to May. Despite the high production of nauplii, Artemia densities declined to 8 l^–1by June and the growing shrimp population grazed down the phytoplankton resource to <1 g Chl a l^–1. With the depleted phytoplankton food resource during the summer, Artemia growth slowed, lipid indices decreased, clutch sizes declined, and females switched primarily to oviparous cyst production. During the summer, there was limited production of ovoviviparous eggs, and limited recruitment of juveniles, probably due to low food. Although oviparous reproduction began in June, more than 90% of the cysts were produced after July when female densities had declined to 1.5 l^–1, but nearly all of them were producing cysts. Estimated cyst production was 650000 m^–2, or 4.54 × 10⁶ kg dry weight for the entire lake. The reported commercial harvest took 21% of the 1994 cyst production. That harvest had little impact on the subsequent year's population, as Artemia densities were ultimately controlled by algal production in the lake.     

An enzymatic route to produce pyruvate from lactate

A bacterial strain of Acinetobacter sp., which was capable of enzymatic production of pyruvate from lactate, was cultured in a 5-l reactor with a basal salt medium. After 14 h of fed-batch fermentation, 9.56 g l^–1 cell concentration in the broth was obtained with 20 g l^–1 (178 mM) sodium lactate and 4 g l^–1 NH₄Cl in the medium; and the biotransformation ability was 2.51 units ml^–1. The cells were harvested from one reactor and then used for pyruvate production from lactate in the same reactor. l-lactate at a concentration about 527 mM was almost stoichiometrically converted to pyruvate in 28 h. After a total 42 h of cell culture and biotransformation, the transformative yield was about 0.72 g g^–1 pyruvate from lactate and the rate of pyruvate production was calculated as 1.33 g l^–1 h^–1 during the process. The results suggested this simple enzymatic production of pyruvate from lactate should be a promising process and may bring a yield higher than that by microbial fermentation. By this process, the recovery of pyruvate from such a simple reaction liquid is relatively easy and inexpensive to perform.     

Influence of inorganic microelements on the production of camptothecin with suspension cultures of <Emphasis Type="Italic">Camptotheca acuminata</Emphasis>

The effects of eight microelements (I^–, BO₃ ^3–, MoO₄ ^2–, Co²⁺, Cu²⁺, Mn²⁺, Fe²⁺, Zn²⁺) on the biosynthesis of camptothecin and the growth of suspension cultures of Camptotheca acuminata were studied. The increase of I^– to 25 M l^–1, Cu²⁺ to 1 M l^–1, Co²⁺ to 2 M l^–1 and MoO₄ ^2– to 10 M l^–1 in Murashige and Skoog (MS) medium resulted in 1.66, 2.84, 2.53 and 2.04 times higher of camptothecin yield than that in standard MS medium respectively. Combined treatment of I^– (25 M l^–1), Cu²⁺ (1 M l^–1), Co²⁺ (2 M l^–1) and MoO₄ ^2– (10 M l^–1) lead to improve cell dry weight, camptothecin content, and camptothecin yield to 30.56 g l^–1, 0.0299%, and 9.15 mg l^–1, respectively, which were 20.2, 208.9 and 273.8% increment respectively when compared with those of control.     

ε-Caprolactam-degradation by Alcaligenes faecalis for bioremediation of wastewater of a nylon-6 production plant

Alcaligenes faecalis G utilized 95–97% of 5–15 g -caprolactam l^–1 in 24–48 h over a pH range of 6–8.5 and at 23–40 °C, without complex nutrient requirement. In the absence of KH₂PO₄ and K₂HPO₄/MgSO₄ in the medium, only 7.6% and 0.2% of 10 g caprolactam l^–1 was utilized, respectively. The chemical oxygen demand (COD) of the wastewater of nylon-6 plant was mainly due to its caprolactam content. A. faecalis G decreased the caprolactam content and COD of the wastewater by 80–90% of the original in spite of the wastewater having higher caprolactam content (3600 mg l^–1) and COD (7700 mg l^–1) than those of any of the previous reports.     

Growth,photosynthesis and photorespiration of Lemna gibba: response to variations in CO2 and O2 concentrations and photon flux density

Dry weight and Relative Growth Rate of Lemna gibba were significantly increased by CO₂ enrichment up to 6000 l CO₂ l^–1. This high CO₂ optimum for growth is probably due to the presence of nonfunctional stomata. The response to high CO₂ was less or absent following four days growth in 2% O₂. The Leaf Area Ratio decreased in response to CO₂ enrichment as a result of an increase in dry weight per frond. Photosynthetic rate was increased by CO₂ enrichment up to 1500 l CO₂ l^–1 during measurement, showing only small increases with further CO₂ enrichment up to 5000 l CO₂ l^–1 at a photon flux density of 210 mol m^–2 s^–1 and small decreases at 2000 mol m^–1 s^–1. The actual rate of photosynthesis of those plants cultivated at high CO₂ levels, however, was less than the air grown plants. The response of photosynthesis to O₂ indicated that the enhancement of growth and photosynthesis by CO₂ enrichment was a result of decreased photorespiration. Plants cultivated in low O₂ produced abnormal morphological features and after a short time showed a reduction in growth.     

N 5-Methyltetrahydromethanopterin: coenzyme M methyltransferase in methanogenic archaebacteria is a membrane protein

An assay is described that allows the direct measurement of the enzyme activity catalyzing the transfer of the methyl group from N ⁵-methyltetrahydromethanopterin (CH₃–H₄MPT) to coenzyme M (H–S–CoM) in methanogenic archaebacteria. With this method the topology, the partial purification, and the catalytic properties of the methyltransferase in methanol- and acetate-grown Methanosarcina barkeri and in H₂/CO₂-grown Methanobacterium thermoautotrophicum were studied. The enzyme activity was found to be associated almost completely with the membrane fraction and to require detergents for solubilization. The transferase activity in methanol-grown M. barkeri was studied in detail. The membrane fraction exhibited a specific activity of CH₃–S–CoM formation from CH₃–H₄MPT (apparent K _m=50 M) and H–S–CoM (apparent K _m=250 M) of approximately 0.6 mol·min^-1·mg protein^-1. For activity the presence of Ti(III) citrate (apparent K _m=15 M) and of ATP (apparent K _m=30 M) were required in catalytic amounts. Ti(III) could be substituted by reduced ferredoxin. ATP could not be substituted by AMP, CTP, GTP, S-adenosylmethionine, or by ATP analogues. The membrane fraction was methylated by CH₃–H₄MPT in the absence of H–S–CoM. This methylation was dependent on Ti(III) and ATP. The methylated membrane fraction catalyzed the methyltransfer from CH₃–H₄MPT to H–S–CoM in the absence of ATP and Ti(III). Demethylation in the presence of H–S–CoM also did not require Ti(III) or ATP. Based on these findings a mechanism for the methyltransfer reaction and for the activation of the enzyme is proposed.Abbreviations H₄MPT tetrahydromethanopterin - CH₃–H₄MPT N ⁵-methyl-H₄MPT - H–S–CoM 2-mercaptoethanesulfonate or coenzyme M - CH₃–S–CoM 2(methylthio)ethanesulfonate or methylcoenzyme M - SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis - DTT dithiothreitol - MOPS morpholinopropanesulfonate - CHAPS 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane-sulfonate - 1 U = 1 mol/min     

Effects of growth temperature on maximal specific growth rate,yield, maintenance,and death rate in glucose-limited continuous culture of the thermophilic Bacillus caldotenax

Summary The influence of temperature on the growth of the theromophilic Bacillus caldotenax was investigated using chemostat techniques and a chemically defined minimal medium. All determined growth constants, that is maximal specific growth rate, yield and maintenance, were temperature dependent. It was striking that the very large maintenance requirement was about 10 times higher than for mesophilic cells under equivalent conditions. A death rate, which was very substantial at optimal and supraoptimal growth temperatures, was estimated by comparing the maintenance for substrate and oxygen. There was no indication for a thermoadaptation as postulated by Haberstich and Zuber (1974).Symbols D Dilution rate (h^–1) - D_c=_max Critical dilution rate (h^–1) - E Temperature characteristic (J mol^–1) - k Organism constant - k_d Death rate coefficient (h^–1) - k_m Maintenance substrate coefficient estimated from M_O (h^–1) - M_O Maintenance respiration, mmol O₂ per g dry biomass and h (mmol g^–1h^–1) - M_O Maintenance respiration, taking k_d into account - m_S Maintenance substrate coefficient, g glucose per g dry biomass and h (h^–1) - OD Optical density at 546 nm - QO₂ Specific O₂-uptake rate (mmol g^–1h^–1) - Q _O2 ^V Specific O₂-uptake rate for viable portion of biomass (mmol g^–1 h^–1) - Q_S Specific glucose uptake rate (h^–1) - Q _S ^V Specific glucose uptake rate for viable portion of biomass (h^–1) - R Gas constant 8.28 J mol^–1K^–1 - S Substrate concentration in reactor (g l^–1) - S_O Influent substrate concentration (g l^–1) - T_max Maximal growth temperature (°C) - T_min Minimal growth temperature (°C) - X Dry biomass (g l^–1) - X_tOt=X Dry biomass containing dead and viable cells - X_v Viable portion of biomass - Y _O ^m Potential yield for O₂ corrected for maintenance respiration (g mol^–1) - Y _S ^m Potential yield for substrate corrected for maintenance requirement, g biomass per g glucose (–) - Specific growth rate (h^–1) - _max Maximal specific growth rate (h^–1)     

Action potential duration and force-frequency relationship in isolated rabbit,guinea pig and rat cardiac muscle

The effect of action potential duration and elevated cytosolic sodium concentration on the forcefrequency relationship in isolated rabbit, guinea pig and rat papillary muscle preparations was studied. Shortening of action potential duration in guinea pig and rabbit from 150–200 ms to values characteristic of rat (20–40 ms), using the K_(ATP) channel activator levkromakalim (15 mol·l^–1), markedly reduced the force of contraction and converted the positive force-frequency relationship into negative one at longer pacing cycle lengths. This conversion was greatly enhanced in the presence of acetylstrophanthidin (0.2–1 mol·l^–1), an inhibitor of the Na-K pump. Acetylstrophanthidin (1 mol·l^–1) alone, however, had no effect on the forcefrequency relationship. Prolongation of action potential duration in rat with inhibitors of cardiac K channels (4-aminopyridine [10 mmol·l^–1] plus tetraethylammonium [2 mmol·l^–1) increased the force of contraction and abolished the negative force-frequency relationship observed in rat at longer pacing-cycle lengths. It is concluded that both action potential duration and cytosolic sodium concentration are major determinants of the force-frequency relationship in mammalian myocardium.Abbreviations AC acetylstrophanthidin - APD action potential duration - APD ₅₀ and APD ₉₀ action potential duration measured at 50% and 90% level of repolarization, respectively - SR sarcoplasmic reticulum     

Sequential kinetic parameter estimation of anaerobic fluidized bed bioreactor using an optimization technique

Mathematical model parameters for the methanogenic degradation of propylene glycol were estimated in a sequential manner by means of an optimization technique. Model parameters determined from an initial experimental data set using one bioreactor were then verified with the results from a second bioreactor. The proposed methodology is a useful tool to obtain model parameters for continuous flow reactors with completely mixed regime. Abbrevations: S – substrate concentration (mg COD l^–1); S _in – influent substrate concentration (mg COD l^–1); D _L – dilution rate (day^–1); – stoichiometric coefficients (ND); nx – number of microbial species (ND); X _S – fixed biomass concentration (mg biomass l^–1); X _L – suspended biomass concentration of (mg biomass l^–1); k _d – decay rate of biomass (day^–1); b _S – specific detachment rate of biofilm (day^–1); – specific growth rate of biomass (day^–1); _m – maximum specific growth rate of biomass (day^–1); K _S – half saturation constant (mg COD l^–1); K _I – inhibition constant (mg COD l^–1).     

Four years continuous record of CH4-exchange between the atmosphere and untreated and limed soil of a N-saturated spruce and beech forest ecosystem in Germany

In order to gain information about seasonal and interannual variations of CH₄-fluxes at a spruce control site, a limed spruce site and a beech site of the Höglwald Forest, Bavaria, Germany, complete annual cycles of CH₄-exchange between the soil and the atmosphere with 2-hourly resolution were followed for 4 consecutive years. The ranges of CH₄ fluxes observed for the different sites were: +12.4 to –69.4 g CH₄ m^–2 h^–1 (spruce control site), +11.7 to –51.4 g CH₄ m^–2 h^–1 (limed spruce site), and –4.4 to –167.3 g CH₄ m^–2 h^–1 (beech site). Lowest rates of atmospheric CH₄-uptake or even a weak net-emission of CH₄ by the soils were observed during winter/spring times, whereas highest rates of CH₄-uptake were always found in summer/spring. Over the entire observation period of 4 years, mean CH₄-uptake rates were –1.82 kg CH₄-C ha^–1 yr^–1 at the spruce control site, –1.31 kg CH₄-C ha^–1 yr^–1 at the limed spruce site, and –4.84 kg CH₄-C ha^–1 yr^–1 at the beech site. The results obtained in this study demonstrate that in view of the huge interannual variations in CH₄-fluxes of approx. 1 kg CH₄-C ha^–1 yr^–1, multiple year measurements of CH₄-fluxes are necessary to accurately characterize the sink strength of temperate forest for atmospheric CH₄. By comparison of CH₄-fluxes measured at the spruce control site and the limed spruce site, a significant negative effect of forest floor liming on CH₄-uptake could be demonstrated. Compared to the spruce stand, the beech stand showed on average approx. 3 times higher rates of atmospheric CH₄-uptake, most likely due to pronounced differences between both sites with regard to the organic layer structure and bulk density of the mineral soil. Regression analysis between CH₄-fluxes and environmental parameters revealed that at all sites the dominating factors regulating temporal variations of CH₄ fluxes were soil moisture and soil temperature. Field measurements of CH₄ concentrations in the soil profile and laboratory measurements of CH₄-oxidation and CH₄-production activity on soil samples taken from different soil depths showed that the CH₄-flux at the Höglwald Forest sites is the net-result of simultaneous occurring production and consumption of CH₄ within the soil. Highest CH₄-oxidation activity was found in the uppermost centimeters of the mineral soil, whereas highest potential CH₄-production activity was found in the organic layer.     

Induced accumulation of triterpenoids in Scutellaria baicalensis suspension cultures using a yeast elicitor

When growth-phase cell suspension cultures of Scutellaria baicalensis were treated with 50 g of yeast elicitor preparation ml^–1, both oleanolic acid and ursolic acid transiently increased in the culture medium rather than in the cells. The maximal triterpenoid concentration was 13.7 mg l^–1 media approx. 35 h after treatment, whereas the maximum concentration was 2.1 mg l^–1 media after about 20 h following treatment with methyl jasmonate. Elicitor treatment also doubled phospholipase A₂ activity (25 pmol mg^–1 min) and the simultaneous treatment of aristolochic acid, a phospholipase A₂ inhibitor, inhibited triterpenoids accumulation as well as phospholipase A₂ activity.     

Organic reduction of tapioca wastewaters using modified RBC

A modified Rotating Biological Contactor (RBC) was used for the treatability studies of synthetic tapioca wastewaters. The RBC used was a four stage laboratory model and the discs were modified by attaching porous nechlon sheets to enhance biofilm area. Synthetic tapioca wastewaters were prepared with influent concentrations from 927 to 3600 mg/l of COD. Three hydraulic loads were used in the range of 0.03 to 0.09 m³·m^–2·d^–1 and the organic loads used were in the range of 28 to 306 g COD· m^–2·d^–1. The percentage COD removal were in the range from 97.4 to 68. RBC was operated at a rotating speed of 18 rpm which was found to be the optimal rotating speed. Biokinetic coefficients based on Kornegay and Hudson models were obtained using linear analysis. Also, a mathematical model was proposed using regression analysis.List of Symbols A m² total surface area of discs - d m active depth of microbial film onany rotating disc - K _s mg ·l^–1 saturation constant - P mg·m^–2·^–1 area capacity - Q l·d^–1 hydraulic flow rate - q m³·m^–2·d^–1 hydraulic loading rate - S ₀ mg·l^–1 influent substrate concentration - S _e mg·l^–1 effluent substrate concentration - w rpm rotational speed - V m³ volume of the reactor - X _f mg·l^–1 active biomass per unit volume ofattached growth - X _s mg·l^–1 active biomass per unit volume ofsuspended growth - X mg·l^–1 active biomass per unit volume - Y _s yield coefficient for attachedgrowth - Y _A yield coefficient for suspendedgrowth - Y yield coefficient, mass of biomass/mass of substrate removed Greek Symbols hr mean hydraulic detention time - (_max)_A d^–1 maximum specific growth rate forattached growth - (_max)_s d^–1 maximum specific growth rate forsuspended growth - _max d^–1 maximum specific growth rate - d^–1 specific growth rate - _v mg·l^–1·hr^–1 maximum volumetric substrateutilization rate coefficient     

Effect of tree distance on N2O and CH4-fluxes from soils in temperate forest ecosystems

Complete annual cycles of N₂O and CH₄ flux in forest soils at a beech and at a spruce site at the Höglwald Forest were followed in 1997 by use of fully automatic measuring systems. In order to test if on a microsite scale differences in the magnitude of trace gas exchange between e.g. areas in direct vicinity of stems and areas in the interstem region at both sites exist, tree chambers and gradient chambers were installed in addition to the already existing interstem chambers at our sites. N₂O fluxes were in a range of –4.6–473.3 g N₂O-N m^–2 h^–1 at the beech site and in a range of –3.7–167.2 g N₂O-N m^–2 h^–1 at the spruce site, respectively. Highest N₂O emissions were observed during and at the end of a prolonged frost period, thereby further supporting previous findings that frost periods are of crucial importance for controlling annual N₂O losses from temperate forests. Fluxes of CH₄ were in a range of +10.4––194.0 g CH₄ m^–2 h^–1 at the beech site and in a range of –4.4––83.5 g CH₄ m^–2 h^–1 at the spruce site. In general, both N₂O-fluxes as well as CH₄-fluxes were higher at the beech site. On a microsite scale, N₂O and CH₄ fluxes at the beech site were highest within the stem area (annual mean: 49.6±3.3 g N₂O-N m^–2 h^–1; –77.2±3.1 g CH₄ m^–2 h^–1), and significantly lower within interstem areas (18.5±1.4 g N₂O-N m^–2 h^–1; –60.2±1.8 g CH₄ m^–2 h^–1). Significantly higher values of total N, C and pH in the organic layer, as well as increased soil moisture, especially in spring, in the stem areas, are likely to contribute to the higher N₂O fluxes within the stem area of the beech. Also for the spruce site, such differences in trace gas fluxes could be demonstrated to exist (mean annual N₂O emission within (a) stem areas: 9.7±0.9 g N₂O-N m^–2 h^–1 and (b) interstem areas: 6.2±0.6 g N₂O-N m^–2 h^–1; mean annual CH₄ uptake within (a) stem areas: –26.1±0.6 g CH₄ m^–2 h^–1 and (b) interstem areas: –38.4±0.8 g CH₄ m^–2 h^–1), though they were not as pronounced as at the beech site.     

Acetone butanol ethanol (ABE) production from concentrated substrate: reduction in substrate inhibition by fed-batch technique and product inhibition by gas stripping

Acetone butanol ethanol (ABE) was produced in an integrated fed-batch fermentation-gas stripping product-recovery system using Clostridium beijerinckii BA101, with H₂ and CO₂ as the carrier gases. This technique was applied in order to eliminate the substrate and product inhibition that normally restricts ABE production and sugar utilization to less than 20 g l^–1 and 60 g l^–1, respectively. In the integrated fed-batch fermentation and product recovery system, solvent productivities were improved to 400% of the control batch fermentation productivities. In a control batch reactor, the culture used 45.4 g glucose l^–1 and produced 17.6 g total solvents l^–1 (yield 0.39 g g^–1, productivity 0.29 g l^–1 h^–1). Using the integrated fermentation-gas stripping product-recovery system with CO₂ and H₂ as carrier gases, we carried out fed-batch fermentation experiments and measured various characteristics of the fermentation, including ABE production, selectivity, yield and productivity. The fed-batch reactor was operated for 201 h. At the end of the fermentation, an unusually high concentration of total acids (8.5 g l^–1) was observed. A total of 500 g glucose was used to produce 232.8 g solvents (77.7 g acetone, 151.7 g butanol, 3.4 g ethanol) in 1 l culture broth. The average solvent yield and productivity were 0.47 g g^–1 and 1.16 g l^–1 h^–1, respectively.     

Formation of phenylethanoid glycosides by Cistanche deserticola callus grown on solid media

The optimal growth of Cistanche deserticola callus and formation of phenylethanoid glycosides (PeG) was at 25°C with light irradiation intensity of 24 mol m^–2 s^–1 on solidified B5 media supplemented with 0.5 mg 6-benzylaminopurine l^–1, 10 mg gibberellin l^–1, 800 mg casein hydrolysate l^–1 and 20 g sucrose l^–1. After 30 d culture, the biomass reached 15.5 g dry wt callus l^–1 medium and its PEG content was 10.7% (w/w). The PeG content was 42%–127% higher than those in explants.