Regeneration of activated charcoal used in decolorization and purification of crude protease from Rhizopus oryzae

Activated charcoal, used for decolorization and purification of crude protease, was regenerated by treatment six times with acetone/water (40:60 v/v), followed by drying. This multistage leaching followed the leaching equation adapted to multistage leaching. The regenerated charcoal was nearly as effective as fresh charcoal in decolorization and purification of crude protease, but only after drying. List of symbols: c ^*, equilibrium solute concentration (w/v) in liquid; c _s, solute concentration in solid phase (w/w); F, interfacial area between solid and liquid phases; k, rate constant for leaching equation; v, volume of liquid per unit weight of adsorbent (solid phase).     

Gas-phase influence on the mixing in a fluidized bed bio-reactor

Summary The liquid and solids mixing in fluidized bed bio-reactors containing particles with a density only slightly higher than water (1100 kg/m³) is generally consistent with the results found in previous studies for reactors with particles of higher density. The liquid mixing can be described by an axial dispersion model for a large variety of conditions while the solids follow the streamlines of the liquid. In the presence of a gas phase the degree of mixing of both the liquid and the solid phase increased. This effect became larger with increasing reactor diameter. In the extrapolation of laboratory data of three phase fluidized bed bio-reactors to pilot plant systems this effect should be taken into account. The liquid and solids mixing may have a substantial effect on overall conversion rates and on possible microbial stratification in the reactor.Nomenclature Bo Bodenstein number v L/D (-) - D _r diameter of the fluidized bed reactor (m) - D ₁ Dispersion coefficient of the liquid phase (m²/s) - D _g dispersion coefficient of the solid phase (m²/s) - E(in) normalized dye concentration function entering the ideally mixed tank reactor (-) - E(t) normalized dye concentration function as measured (-) - L length of the axial dispersed reactor (m) - t time after dye injection (s) - t _m time constant for microbial selection (s) - t _s solid mixing time constant (s) - t time interval in which a particle migrates within the bed (s) - v _t superficial gas velocity (m/s) - v _g superficial liquid velocity (m/s) - z migration distance of a particle in the bed (m) - ₁ in situ growth rate of a dominant organism (s^-1) - ₂ in situ growth rate of a recessive organism (s^-1) - average residence time in the axial dispersed reactor (s) - _t average residence time in the ideally mixed tank reactor (s)     

Study of the liquid circulation velocity in external-loop airlift bioreactors

Liquid circulation velocity was studied in externalloop air-lift bioreactors of laboratory and pilot scale, respectively for different gas input rates, downcomer-to-riser cross-sectional area ratio, A _D/A_R and liquid phase apparent viscosities.It was found that, up to a gas superficial velocity in the riser v _SGR 0.04 m/s the dependency of v _SLR on v _SGR is in the following form: v _SLR = a v _SGR ^b , with the exponent b being 0.40. Over this value of v _SGR, only a small increase in liquid superficial velocity, v _SLR is produced by an increase in v _SGR. A _D/A_R ratio affects the liquid superficial velocity due to the resistance in flow and overall friction.For non-Newtonian viscous liquids, the circulation liquid velocity in the riser section of the pilot external-loop airlift bioreactor is shown to be dependent mainly on the downcomer-to-riser cross-sectional area ratio, A _D/A_R, the effective (apparent) liquid viscosity, _eff and the superficial gas velocity, v _SGR.The equation proposed by Popovic and Robinson [11] was fitted well, with an error of ± 20%.List of Symbols A _D m² downcomer cross-sectional area - A _Rm² riser cross-sectional area - a = coefficient in Eq. (7) - b = exponent in Eq. (7) - c _s m^–1 Coefficient in Eq. (3) - D _D m downcomer diameter - D _R m riser diameter - g m²/s gravitational acceleration - H _D m dispersion height - H _L m ungassed liquid height - K Pa s ⁿ consistency index - K _B = friction factor at the bioreactor bottom - K _F = friction factor - K _T = friction factor at the bioreactor top - V _L m³ liquid volume in the bioreactor - V _D m³ liquid volume in downcomer - V _R m³ liquid volume in riser - v _LDm/s downcomer linear liquid velocity - v _LR m/s riser linear liquid velocity - v _SGR m/s riser superficial liquid velocity - v _SLR m/s riser superficial liquid velocity - s^–1 shear rate - _GD = downcomer gas holdup - _GR = riser gas holdup - _eff Pa s effective (apparent) viscosity - Pa shear stress The authors wish to thank Mrs. Rodica Roman for the help in experimental data collection and to Dr. Stefanluca for the financial support.     

Plant regeneration from cotyledon protoplasts of Xinjiang muskmelon

Cotyledon protoplasts were isolated from seedlings of Xinjiang muskelon (Cucumis melo var.saccharinus) grown under sterile conditions and cultured in modified Miller medium. High frequency division of the protoplast-derived cells was observed. Agarose bead culture with B₆S₃ tobacco crown gall nurse cells was found most suitable for the cotyledon protoplasts when compared with other culture methods. Intact plants were regenerated from the protocalli by a two-step culture procedure with liquid and then solid media.Abbreviations BAP 6-benzylaminopurine - B₆S₃ crown gall tumor cells of tobacco - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - MES 2(Nmorpholino) ethanesulfonic acid - MS Murashige and Skoog medium(1962) - NAA -naphthaleneacetic acid - N6 Zhu et al. medium (1975)     

Characterization of the physical properties of yeast flocs

Summary Physical characteristics, namely floc density function, floc size distribution, and relative floc strength, of a number of flocculent yeast types were measured. A straight-line relationship was found to exist between log values of size and density for the yeasts examined. Each yeast type had coefficients from this relationship which could be used to interpret settling behaviour. Indices of relative floc strength were also obtained and together with the floc density function allowed fuller interpretation of yeast settling than with simpler theories.Symbols a constant (g·cm^-3) - B ₂/B ₁ floc binding strength of floc₂ relative to floc₁ - d _f floc diameter (cm) - d _i image diameter on print (cm) - d _max maximum floc diameter (cm) - f _d Ploc effective density (g·cm^-3) - g gravitational constant (981 cm·s^-1) - K _p constant (-) - R _l rate of enlargement on film - R ₂ rate of enlargement on print - S _s density of suspending liquid phase (g·cm^-3) - S _f density of solid (floc) phase (g·cm^-3) - U _t terminal settling velocity (cm·s^-1) - u liquid viscosity (g·cm^-3·^-1)     

Convenient experimental determination of adsorption isotherms in a Hydrophobic Interaction Chromatography system

Summary HPLC was combined with a packable microbore guard column to obtain the adsorption isotherm of lysozyme in a Hydrophobic Interaction Chromatography system. The equipment configuration enabled isotherm determination of the protein on a relatively low pressure chromatographic media (TosoHaas 650M Phenyl).Notation C_m,i is the mobile phase concentration of protein. (M/L³ _(liquid)) - C_m,0 =0 - C_s,i is the stationary phase concentration of protein. It is the concentration of protein on the chromatographic media. (M/L³ _(solid)) - C_s,0 =0 - M,L is the dimensions mass and length - V_r,i is the retention volume of the peak front that corresponds to a mobile phase protein on the concentration C_m,i. (L³ _(liquid)) - i i is a counter that is used to keep track of C_m, C_s, and V_r.For example, i=1 in the term C_m,i denotes the first, and lowest, mobile phase protein concentrations are described by higher values of i. - V_d is the system dead volume. It consists of all of the system volume that the mobile phase "sees" or contacts, includingchromatographic media interparticle and pore volume. (L³ _liquid) - V_s the stationary phase volume. V_s is the nonporous bead volume. For porous beads, V_s is the bead volume - the porevolume. (L³ _(solid)) - V_e is the empty column volume. (L³ _liquid) - V_m is the packed column mobile phase volume and consists of the pore volume and the excluded volume. (L³ _(liquid)) - V_{e system} is the empty column system volume. (L³ _(liquid)) - V_frit the volume of mobile phase that fills the column frits. (L³ _(liquid)) - V_woc the system volume without the column connected. (L³ _(liquid))     

The behaviour of yeast flocs in hindered settling and fluidized bed regimes

Summary Sedimentation and fluidization of yeast flocs were found to be non-synonymous processes. The analysis of Richardson and Zaki (1954) was found not to hold when applied to yeast flocs in both regimes. Partial support and channelling were implicated in the deviations from idela behaviour. Other factors responsible for the behaviour of yeast flocs in these regimes are discussed.Symbols D bed height (cm) - g gravitational constant (981 cm·s^-1) - n constant (-) - R retardation factor (s) - S constant (-) - v liquid/particle velocity (cm·s^-1) - V _o particle terminal velocity (cm·s^-1) - bed voidage (-)     

Potential of solid state fermentation for production of L(+)-lactic acid by Rhizopus oryzae

Production of l(+)-lactic acid by Rhizopus oryzae NRRL 395 was studied in solid medium on sugar-cane bagasse impregnated with a nutrient solution containing glucose and CaCO₃. A comparative study was undertaken in submerged and solid-state cultures. The optimal concentrations in glucose were 120 g/l in liquid culture and 180 g/l in solid-state fermentation corresponding to production of l(+)-lactic acid of 93.8 and 137.0 g/l, respectively. The productivity was 1.38 g/l per hour in liquid medium and 1.43 g/l per hour in solid medium. However, the fermentation yield was about 77% whatever the medium. These figures are significant for l(+)-lactic acid production.     

Citric acid production from sugar cane molasses by 2-deoxyglucose-resistant mutant strain ofAspergillus niger

Citric acid production from sugar cane molasses byAspergillus niger NIAB 280 was studied in a batch cultivation process. A maximum of 90 g/L total sugar was utilized in citric acid production medium. From the parental strainA. niger, mutant strains showing resistance to 2-deoxyglucose in Vogal's medium containing molasses as a carbon source were induced by γ-irradiation. Among the new series of mutant strains, strain RP7 produced 120 g/L while the parental strain produced 80 g/L citric acid (1.5-fold improvement) from 150 g/L of molasses sugars. The period of citric acid production was shortened from 10 d for the wild-type strain to 6–7 d for the mutant strain. The efficiency of substrate uptake rate with respect to total volume substrate consumption rate,Q _s (g per L per h) and specific substrate consumption rate,q _s (g substrate per g cells per h) revealed that the mutant grew faster than its parent. This indicated that the selected mutant is insensitive to catabolite repression by higher concentrations of sugars for citric acid production. With respect to the product yield coefficient (Y _p/x), volume productivity (Q _p) and specific product yields (q _p), the mutant strain is significantly (p≤0.05) improved over the parental strain.     

Ultrastructural study of different types of callus from leaf expiants ofAesculus hippocastanum L.

Summary The cell ultrastructure in three types of callus obtained from leaf explants ofAesculus hippocastanum L. has been studied. Remarkable differences have been shown between the cells of the forerunner E₁ callus and those of the callus arising from it, according to the culture conditions.The peculiar characteristics of E₁ are the scarcity of intercellular spaces and the occurrence of autophagic vacuoles in the cells.An embryogenic friable callus (E₂) is formed in time when E₁ is maintained on solid culture medium. The E₂ cells show cytological features typical of a higher metabolic level and contain starch. Diffused middle lamella digestion leads to the detachment of small embryogenic cell aggregates consisting of vacuolated parenchymatous-like cells and small meristematic cells which may be regarded as embryoids initials.Shaking E₁ in the same liquid medium and subsequent culture on solid medium lead to the differentiation of a non-embryogenic callus (NE), whose cells are very large and highly vacuolated, devoid of starch and with organelle-rich cytoplasm. The NE callus shows a high degree of growth, but does not attain embryogenic competence in time.Abbreviations c cell - cr crystal - cw cell wall - d dictyosome - er endoplasmic reticulum - m mitochondrion - mb microbody - n nucleus - p plastid - s starch - v vacuole     

A submerged culture system for rapid micropropagation of the commercially important aquarium plant, ‘Amazon sword’ (Echinodorus ‘Indian Red’)

Echinodorus ‘Indian Red’ is an underwater plant, used worldwide for aquarium ornamentation. An efficient method for in vitro propagation and plantlet acclimatization of this popular aquarium plant was standardized. Surface-disinfected shoot-tips were cultured in submerged conditions in a solid–liquid bilayer medium, consisting of an upper, liquid layer (sterile distilled water) and a lower, solid layer Murashige and Skoog (MS) basal medium supplemented with 3.0% (w/v) sucrose, 0.8% (w/v) agar-agar, and plant growth regulators (PGRs) in different combinations and concentrations. The combination of 2.5 mg L⁻¹ 6-benzylaminopurine and 1.0 mg L⁻¹ α-naphthaleneacetic acid improved the multiplication rate to a maximum of 26.8 ± 0.51 shoots per explant after 60 d of culture. The number of multiplied shoots increased with each regeneration cycle, thus from only 26.8 ± 0.51 shoots per explant (first regeneration cycle), this number increased to 33.5 ± 0.58 (second regeneration cycle), and to 38.3 ± 0.62 for the third regeneration cycle with the same medium composition. The highest number of roots (8.3 ± 0.28) per shoot was induced in the presence of 1.0 mg L⁻¹ indole-3-butyric acid, but further growth of these roots was stunted. The best rooting was achieved on PGR-free ½-strength MS medium, where 6.1 ± 0.21 roots per shoot were induced with 5.8 ± 0.35 cm length after 30 d of culture. The regenerated plantlets were successfully acclimatized to submerged underwater conditions, with 100% survival rate. The present protocol is suitable for the commercial propagation of Echinodorus ‘Indian Red’ for aquarium-industries.

     

Flow characteristics of a pilot-scale airlift fermentor

The effects of aeration on the flow characteristics of water in a glass pilot-scale airlift fermentor have been examined. The 55-L capacity fermentor consisted of a 15.2-cm-i.d. riser column with a 5.1-cm-i.d. downcomer tube. It was found that the average bubble size diminished with increased aeration. Typically, average bubble sizes ranged from 4.32 mm at a superficial gas velocity of 0.64 cm/s to 1.92 mm at 10.3 cm/s. A gas holdup of 0.19 was attained with superficial gas velocities (v_s) on the order of 10 cm/s, indicating the highly gassed nature of the fluid in the riser section of the fermentor. Circulation velocities of markers placed in the fermentor decreased with increasing aeration rates due to increased turbulence and axial liquid back mixing within the riser section. Actual volumetric liquid circulation rates remained relatively constant (0.36–0.49 L/s) for values of (v_s) up to 10 cm/s. Based on theoretical calculations, the ascending velocity of bubbles in a swarm reached 54 cm/s in the range of (v_s) values studied.     

Improvement of Aconitum napellus micropropagation by liquid culture on floating membrane rafts

An efficient method was developed using floating membrane rafts (Liferaft) for the micropropagation of Aconitum napellus (Ranunculaceae), a cut flower crop with a low natural propagation rate. This was achieved by introducing shoot tips into culture on Murashige and Skoog's (1962) solid medium, or liquid medium-supported rafts, supplemented by different levels of benzyl adenine (BA). Optimum shoot proliferation on solid medium required 4mg/l BA, whereas for expiants supported on rafts optimal proliferation was achieved at 0.25mg/l BA. Maximum shoot proliferation was found using the floating rafts (propagation ratio of 4.2 per month), 45% higher than the maximum value on solid medium. A similar value could be obtained on solid medium after a period of 2 months. The optimal response to BA was similar for fresh weight gain and shoot length. Growth in a shallow layer of liquid in shake flasks gives a similar shoot multiplication rate to that on floating rafts; however, submerged leaves brown and die.Abbreviations BA 6-benzylaminopurine - GA₃ Gibberellic acid - IBA indole-3-butyric acid - IAA indole-3-acetic acid - NAA naphthalene acetic acid     

Theoretical analysis of the baker's yeast production: An experimental verification at a laboratory scale

The scale-down procedure can be used to optimize and scale up fermentation processes. The first step in this procedure, a theoretical analysis of the process at a large scale, must give information about the regime, or bottle necks, ruling the process. In order to verify the theoretical results the process analysis has been applied to the fed-batch baker's yeast production at a laboratory scale. The results of this analysis are compared with results from fed-batch experiments. It was concluded that if only one mechanism is ruling the process, for instance mass transfer, the results of the analysis are quite clear. If more than one mechanism is important, for example mass transfer and liquid mixing, additional knowledge is needed to predict the behaviour of the process.Concerning the baker's yeast production, it was concluded that if oxygen limitation occurs, liquid mixing is of little importance.List of Symbols C kg/m³ concentration - C ^* kg/m³ saturation concentration - D m diameter - D _E m²/s effective dispersion coefficient - d m holes of the sparger - F _sm³/s substrate flow to the fermentor - g m/s² gravitational acceleration - H m height - k _La s^–1 volumetric mass transfer coefficient based on the liquid volume - L m length - m _skg/(kg·s) maintenance coefficient - OTR kg/(m³·s) oxygen transfer rate - OUR kg/(m³·s) oxygen uptake rate - r kg/(m³·s) reaction rate - t s time - V m³ volume - v m/s velocity - v _sm/s superficial gas flow rate - y _ijkg/kg yield of componentj oni - s^–1 specific growth rate - s time constant - _gm³/s gas flow rate Indices 0 value att=0 - cir liquid circulation - e ethanol - f feed concentration - g gas phase - in flow going to the fermentor - l liquid phase - m mixing - mt mass transfer - o, O₂ oxygen - oc oxygen consumption - out flow coming out the fermentor - s substrate - sa substrate addition - sc substrate consumption - x biomass     

The effect of light levels on daily patterns of chlorophyll fluorescence and organic acid accumulation in the tropical CAM treeClusia hilariana

Sandy plains are characteristic of the coastal region of Brazil. We investigated the diel patterns of changes in organic acid levels, leaf conductance and chlorophylla fluorescence for sun-exposed and shaded plants ofClusia hilariana, one of the dominant woody species in the sandy coastal plains of northern Rio de Janeiro state. Both exposed and shaded plants showed a typical CAM pattern with considerable diel oscillations in organic acid levels. The degradation of both malic and citric acids during the midday stomatal closure period could lead to potential CO₂ fixation rates of 28 mol m^-2 s^-1 in exposed leaves. Moreover, exposed leaves exhibited large increases in total non-photochemical quenching (q_N) accompanied by a substantial decrease in effective quantum yield during the course of the day. However, these potential high rates of CO₂ fixation and the increases inqn of exposed plants were not enough to maintain the primary electron acceptor of photosystem II (q_A) in a low reduction state, similar to that of shaded plants. As a result, there was a moderate increase in the reduction state of q_A throughout the day. Most of the decline in photochemical efficiency of exposed leaves ofC. hilariana was reversible, as evidenced by the high levels of pre-dawn potential quantum yields (F_v/F_m) and their rapid recovery after sunset. However, the depletion of the organic acid pool in the afternoon resulted in an accentuated subsequent drop in F_v/F_m, suggesting that prolonged periods of water stress accompanied by high irradiance levels may expose plants ofC. hilariana in unprotected habitats to the danger of photoinhibition.     

The kinetic basis of the role of Ca++ ions for higher yield of citric acid in a repeated-batch cultivation system

The present investigation deals with role of Ca⁺⁺ ions in increasing the yield of citric acid in a repeated-batch cultivation system (working volume 9-1) and its kinetic basis. Five different hyper-producing strains of Aspergillus niger were evaluated for citric acid production using clarified cane-molasses as basal substrate. Among the cultures, NG^GCB101 (developed by u.v./chemical mutation in our labs) gave maximum production of citric acid i.e., 87.98 g/1, 6 days after mycelial inoculation. The addition of CaCl₂ to the culture medium promoted the formation of small rounded fluffy pellets (1.55 mm, diameter), which were desirable for citric acid productivity. CaCl₂ at a level of 2.0 M, added during inoculation time, was optimized for commercial exploitation of molasses. During repeated-batch culturing, a yield of citric acid monohydrate of 128.68 g/1 was obtained when the sampling vs. substrate feeding was maintained at 4-1 (44.50% working volume). The incubation period was reduced from 6 to only 2 days. The values of kinetic parameters such as substrate consumption and product formation rates revealed the hyperproducibility of citric acid by the selected Aspergillus niger NG^GCB101 (LSD = 0.456a, HS). Case studies are highly economical because of higher yield of product, lower energy consumption and the use of raw substrate without any additional supplementation.     

A High Performance Fermentation Process for Citric Acid Production by Aspergillus niger NGGCB-101, Using Vermiculite as an Additive in a Stirred Bioreactor

The present study describes the use of vermiculite for enhanced citric acid productivity by a mutant strain of Aspergillus niger NG^GCB-101 in a stirred bioreactor of 15.0 l capacity. The maximum amount of citric acid (96.10 g/l) was obtained with the control 144 h after mycelial inoculation. To enhance citric acid production, varying levels of vermiculite were added as an additive into the fermentation medium. The best results were observed when 0.20 g/l vermiculite was added into the medium 24 h after inoculation resulting in the production of 146.88 g citric acid monohydrate/l. The dry cell mass and residual sugar were 11.75 and 55.90 g/l, respectively. Mixed mycelial pellets (1.08–1.28 mm, dia) were observed in the fermented culture broth. When the culture grown at different vermiculite levels was monitored for Q _p , Q _s and q _p , there was a significant enhancement (P 0.05) in these variables over the control (vermiculite-free). Based on these results, it is concluded that vermiculite might affect mycelial morphology and subsequent TCA cycle performance to improve carbon source utilization by the mould, basic parameters for high performance citric acid fermentation.     

Effects of Micropropagation Conditions of Rose Shootlets on Chlorophyll Fluorescence

Rosa hybrida plantlets were rooted on solid sucrosed medium (MS) under an irradiance (PPFD) of 45 μmol m_-2 s_-1 or on liquid hydroponic solution (MH) at 100 μmol m_-2 s_-1. Then all plantlets were acclimated without sucrose under 100 μmol m_-2 s_-1 PPFD. After 7 d in rooting stage, the ratio of variable over maximal chlorophyll fluorescence (F_v/F_m) was significantly higher for plants grown in MH than in MS and hence the higher irradiance at this stage of growth had no photoinhibitory effect. The radiant energy was used by the photochemical process and also by photoprotective mechanisms of photosystem 2, expressed by increases in the rates of electron flux, net photosynthesis, and non-photochemical quenching. This effect on F_v/F_m was maintained during three weeks in acclimation phase. The resistance of plantlets increased as new leaves formed, and after six weeks in acclimation, there was no difference between the two conditions. The study under higher irradiance (100, 150, or 300 μmol m_-2 s_-1) indicated that photoinhibition might take place at 300 μmol m_-2 s_-1 whatever the growth conditions. This revised version was published online in September 2006 with corrections to the Cover Date.     

The growth conditions of cephalosporium eichhorniae for the conversion of starch substrates to protein

The protein synthesis by Cephalosporium eichhorniae on substrates containing starch was evaluated at different pH, moisture content, ammonium sulphate and potassium dihydrogen phosphate supplementation. The optimum pH level was about 4.2 and the moisture content 65%. The optimum level of supplementation of medium containing sweet potato substrate with (NH₄)₂SO₄ was smaller than that for cassava substrate (5.2 g/l < 6.7 g/l in submerged culture, and 4.0% < 5.2% in solid state fermentation). The total crude protein yields were about 7.6 g/l for submerged cultures and 12% DM for solid state fermentations.     

Cardiac output and stroke volume in swimming harbor seals

Summary Cardiac output was measured by the thermodilution method in three young harbor seals, at rest and while swimming up to the maximum effort for which they could be trained. Stroke volume was determined by counting heart rate simultaneously with determination of cardiac output. Cardiac outputs varied widely between surface breathing (7.8 ml · kg^–1 · s^–1) and breath-holding while swimming under water (1.8 ml · kg^–1 · s^–1). Stroke volume while at the surface was almost twice the volume white submerged. Surface cardiac output was always near maximal despite work effort, whereas submerged cardiac output gradually increased at higher work efforts. The cardiovascular performance of seals at the maximum MO₂ we could induce from them is equivalent to that of the domestic goat.Abbreviations CO Cardiac output - HR Heart rate - SV Stroke volume - MO ₂ Metabolic rate - FS Forced sumersion - V Velocity - C _DF Frontal drag coefficient - CV Cardiovascular Present address: Institute of Marine Science, University of Alaska, Fairbanks, AK, USA