Animal cell culture in stirred bioreactors: Observations on scale-up

Scale-up of stirred tank bioreactors from 0.02 m³ to 0.3 m³ commercial plant is discussed for hybridoma suspension culture. Schemes for dissolved oxygen control with sparged air in serum containing media are described as well as mechanical breakage of foam in small and large bioreactors. Porous metal spargers (180?200×10^?6 m) are found to produce foams which are hard to control. Aeration with larger (> 0.001 m) multihole spargers is recommended. Combined cell damage due to foam formation and control, and possible damage at mechanical seals or submerged bearings, are found to have no measurable effect on cell growth relative to roller bottle production. Hybridomas are shown to withstand significant impeller tip speed (> 1 ms^?1) and fluid turbulence as evidenced by impeller Reynolds numbers in excess of 10⁵. The size of the energy dissipating terminal eddies is calculated to be > 10-fold that of the hybridoma cells. Specific fluid turnover rate is employed as the scale-up criterion.     

Determination of bacterial cell number and cell volume by means of flow cytometry, transmission electron microscopy, and epifluorescence microscopy

Comparative measurements of bacterial total counts and volumes of flow cytometry (FCM), transmission electron (TEM), and epifluorescence microscopy (EFM), were undertaken during a four week mesocosm experiment. Total counts of bacteria measured by TEM, EFM, and FCM were in the range of 1 · 10⁶−6 cells ml⁻¹, 1 · 10⁶−3 · 10¹⁶ cells ml⁻¹, and 5 · 10⁵ cells ml⁻¹ respectively. The mean volume of the bacterial community, measured by means of EFM and TEM, increased from 0.12–0.15 μm³ at the start of the experiment to 0.39–0.53 μm³ at the end. Generally, there was good agreement between the two methods and regression analyses gave r = 0.87 (p < < 0.01) for cell volume and r = 0.97 (p < < 0.01) for cell number. DAPI stained bacteria with volumes less than 0.2 μm³ were not detected by flow cytometry and these were generally an order of magnitude lower than counts made by TEM and EFM. For samples where the mean bacterial cell volume was longer than 0.3 μm³, all three methods were in agreement both with respect to counts and volume estimates.     

Biotreatment of swine manure by intensive lagooning during winter

The intent of the proposed system was to develop an in situ biotreatment system for swine manure that should involved minimum handling by the farmer. A demonstration plant has been installed in the Paris region. The intensive lagooning system consists of algal ponds, daphnid ponds and a polishing fish pond working in series with a total area of 2100 m² and a total volume of 3600 m³.

The performance of the system was evaluated under winter conditions with raw decanted swine manure by measuring N-NH₄⁺ and P-PO₄³⁻ removal, pH, temperature, water oxygen, algal biomass and daphnid productions.

Results showed that low temperatures (<5°C) did not allow any significant biomass production (0·41–0·68 g dry mass/m² d). Ammonia-nitrogen, mostly stripped (98% lost by stripping), and phosphate removals were small. However, as soon as the temperature increased in spring (March), ammonia-nitrogen removal was improved with a large contribution (71%) due to stripping and the algal productivity increased to ≈ 3·5 g dry mass/m² d; CO₂ appeared to be a limiting factor.

Ammonia-nitrogen, nitrate, nitrite and orthophosphate showed marked variations that could be correlated with manure overdoses.     

Rate of electron transfer between plastocyanin, cytochrome ƒ, related proteins and artificial redox reagents in solution

The rate of electron transfer between reduced cytochrome ƒ and plastocyanin (both purified from parsley) has been measured as k = 3.6 · 10⁷ M⁻¹ · s⁻¹, at 298 °K and pH 7.0, with activation parameters ΔH^‡ = 44 kJ · mole⁻¹ and ΔS^‡ = +46 J · mole⁻¹ · °K⁻¹. Replacement of cytochrome ƒ with red algal cytochrome c-553, Pseudomonas cytochrome c-551 and mammalian cytochrome c gave rates at least 30 times slower: k = 5 · 10⁵, 7.5 · 10⁵ and 1.0 · 10⁶ M⁻¹ · s⁻¹, respectively.

Similar measurements made with azurin instead of plastocyanin gave k = 6 · 10⁶ and approx. 2 · 10⁷ M⁻¹ · s⁻¹ for reaction of reduced azurin with cytochrome ƒ and algal cytochrome respectively.

Rate constants of 115 and 80 M⁻¹ · s⁻¹ were found for reduction of plastocyanin by ascorbate and hydroquinone at 298 °K and pH 7.0. The rate constants for the oxidation of plastocyanin, cytochrome ƒ, Pseudomonas cytochrome c-551 and red algal cytochrome c-553 by ferricyanide were found to be between 3 · 10⁴ and 8 · 10⁴ M⁻¹ · s⁻¹.

The results are discussed in relation to photosynthetic electron transport.     

Influence of phosphorus concentration on the growth kinetics and stoichiometry of the microalga Scenedesmus obliquus

The growth of the freshwater microalga Scenedesmus obliquus was studied at 30°C in a mineral culture medium with phosphorus concentrations of between 0 and 372 μ . The values for the specific growth rates, between and , fitted a semistructured substrate-limitation model with μ_m1 = 0·0466 h⁻¹, μ_m2 = 0·0256 h⁻¹ and . The specific uptake rate of phosphorus reached a maximum value of q_Sm1 = 658·01 × 10⁻⁴ μmol P mg⁻¹ biomass h⁻¹.     

Power consumption of three impeller combinations in mixing Xanthan fermentation broths

Xanthan gum fermentation represents a good model for the study of the mixing of rheologically complex culture broths. Most of the previous work on power consumption dealt with ‘standard’, single impellers and used model fluids to simulate xanthan broths. This work describes the characterization of three dual-impeller combinations (D/T = 0·53) for the mixing of dehydrated—reconstituted fermentation broths of Xanthomonas campestris that had matched rheology to the actual broths. The bottom impeller was a Rushton turbine (RT) and the top impeller was another RT, a 45° pitched blade turbine (PT) or an A-310 Lightnin mixer (A310). The experiments were carried out in a tank of 0·0094 m³ working volume equipped with an air bearing dynamometer. The power was measured in a wide range of xanthan concentrations (5–40 kg m⁻³) in aerated (0·25, 0·5 and 1·0 vvm) and unaerated conditions. Unaerated power number (Po) vs. Reynolds number (Re) curves showed similar trends for the three combinations. Exponents close to −1 were obtained in the laminar region. A minimum in Po (Po_min) occurred at Re = 30–40, then increasing to a plateau value which was evident at Re> 200. In the transition region Po_min values were 4·3 (RT and RT), 3·6 (RT and PT) and 2·4 (RT and A310). The aerated power data for (RT and PT) and (RT and A-310) showed higher torque instabilities than the dual RT combinations at higher xanthan concentrations. The higher the xanthan concentrations, the higher the drop in power and the less important the effect of the aeration rate. Among the combinations tested, when using Rushton turbines, the well-mixed ‘cavern’ reached the tank wall (i.e., fluid motion was observed) at the lowest volumetric power input. High     

Mercury Removal from Air by a Fiber-Based Bioreactor

A partial removal of metallic mercury from air by fiber-based trickle-bed bioreactors was observed. Up to 50 to 65% of the inlet mercury concentrations of 35 to 70 µg/m³ were removed by immobilized live Pseudomonas bacteria for up to 275 hours at a residence time of 1 min. Ninety to 125% of the adsorbed mercury was recovered by a direct assay after dismantling the bioreactors, thus confirming that the observed mercury removal was due to its adsorption by biomass rather than wet scrubbing followed by evaporation. However, mercury removal at a lower inlet concentration (23 µg/m³) was negligible, with a poor material balance. The adsorbed mercury at higher inlet concentrations was not removed from the biomass by a 2-week washing after conclusion of the mercury adsorption experiment, which indicates a strong mercury binding by bacteria. The volatile organic compound removal efficiency was not affected by the presence of up to 70 µg/m³ of metallic mercury in the air.     

Engineering characterisation of a single well from 24-well and 96-well microtitre plates

The detailed engineering characterisation of shaken microtitre-plate bioreactors will enhance our understanding of microbial and mammalian cell culture in these geometries and will provide guidance on the scale-up of microwell results to laboratory and pilot scale stirred bioreactors. In this work computational fluid dynamics (CFD) is employed to provide a detailed characterisation of fluid mixing, energy dissipation rate and mass transfer in single well bioreactors from deep square 24-well and 96-well microtitre plates. The numerical predictions are generally found to be in good agreement with experimental observation of the fluid motion and measured values of the key engineering parameters. The CFD simulations have shown that liquid mixing is more intensive in 96-well than in 24-well bioreactors due to a significant axial component to the fluid velocity. Liquid motion is strongly dependent on the orbital shaking amplitude which generally has a greater impact than the shaking frequency. Average power consumptions of 70–100 W m⁻³ and 500–1000 W m⁻³, and overall mass transfer coefficient, k_La, values of 0.005–0.028 s⁻¹ and 0.056–0.10 s⁻¹ were obtained for 24-well and 96-well bioreactors respectively at an orbital shaking amplitude of 3 mm and shaking frequencies ranging from 500 rpm to 1500 rpm. The distribution of energy dissipation rates within each bioreactor showed these to be greatest at the walls of the well for both geometries. Batch culture kinetics of E. coli DH5 showed similar maximum specific growth rates and final biomass yields in shaken 24-well and shake flask bioreactors and in stirred miniature and 20 L bioreactors at matched k_La values. The CFD simulations thus give new insights into the local and overall engineering properties of microwell bioreactor geometries and further support their use as high throughput tools for the study and optimisation of microbial and mammalian cell culture kinetics at this scale.     

A convenient electrochemical preparation of reduced methyl viologen and a kinetic study of the reaction with oxygen using an anaerobic stopped-flow apparatus

1. Single reduced methyl viologen (MV^.+) acts as an electron donor in a number of enzyme systems. The large changes in extinction coefficient upon oxidation (λ_max 600 nm; MV^.+, = 1.3 · 10⁴ M⁻¹ · cm⁻¹; oxidised form of methyl viologen (MV²⁺), = 0.0) make it ideally suited to kinetic studies of electron transfer reactions using stopped-flow and standard spectrophotometric techniques.

2. A convenient electrochemical preparation of large amounts of MV^.+ has been developed.

3. A commercial stopped-flow apparatus was modified in order to obtain a high degree of anaerobicity.

4. The reaction of MV^.+ with O₂ produced H₂O₂ (k > 5 · 10⁶ M⁻¹ · s⁻¹, pH 7.5, 25 °C). H₂O₂ subsequently reacted with excess MV^.+ (k = 2.3 · 10³ M⁻¹ · s⁻¹, pH 7.5, 25 °C) to produce water. The kinetics of this reaction were complex and have only been interpreted over a limited range of concentrations.

5. The results support the theory that the herbicidal action of methyl viologen (Paraquat, Gramoxone) is due to H₂O₂ (or radicals derived from H₂O₂) induced damage of plant cell membrane.     

Thermodynamics of the disproportionation of adenosine 5'-diphosphate to adenosine 5'-triphosphate and adenosine 5'-monophosphate, II. Experimental data

High-pressure liquid-chromatography and microcalorimetry have been used to determine equilibrium constants and enthalpies of reaction for the disproportionation reaction of adenosine 5′-diphosphate (ADP) to adenosine 5′-triphosphate (ATP) andadenosine 5′-monophosphate (AMP). Adenylate kinase was used to catalyze this reaction. The measurements were carried out over the temperature range 286 to 311 K, at ionic strengths varying from 0.06 to 0.33 mol kg⁻¹, over the pH range 6.04 to 8.87, and over the pMg range 2.22 to 7.16, where pMg = -log a(Mg²⁺). The equilibrium model developed by Goldberg and Tewari (see the previous paper in this issue) was used for the analysis of the measurements. Thus, for the reference reaction: 2 ADp³⁻ (ao) AMp²⁻ (ao)+ ATp⁻ (ao), K° = 0.225 ± 0.010, ΔG° = 3.70 +- 0.11 kJ mol ⁻¹, ΔH° = −1.5 ± 1. 5 kJ mol ⁻¹, °S ° = −17 ± 5 J mol⁻¹ K⁻¹, and ACP_p°≈ = −46 J mo1l⁻¹ K⁻¹ at 298.15 K and 0.1 MPa. These results and the thermodynamic parameters for the auxiliary equilibria in solution have been used to model the thermodynamics of the disproportionation reaction over a wide range of temperature, pH, ionic strength, and magnesium ion morality. Under approximately physiological conditions (311.15 K, pH 6.94, [Mg²⁺] = 1.35 × 10⁻³ mol kg⁻¹, and I = 0.23 mol kg⁻¹) the apparent equilibrium constant (K_A′ = m(ΣAMP)m(ΣATP)/[ m(ΣADP)]²) for the overall disproportionation reaction is equal to 0.93 ± 0.02. Thermodynamic data on the disproportionation reaction and literature values for this apparent equilibrium constant in human red blood cells are used to calculate a morality of 1.94 × 10⁻⁴ mol kg⁻¹ for free magnesium ion in human red blood cells. The results are also discussed in relation to thermochemical cycles and compared with data on the hydrolysis of the guanosine phosphates.     

Performance evaluation of a 1 m modified, fixed-dome Deenbandhu biogas plant under hilly conditions

The performance of a 1 m³ daily gas-production-capacity, modified, Deenbandhu biogas plant was evaluated under hilly conditions. The modified plant had a digester volume of 2·65 m³ with 55 days hydraulic retention time (HRT) as against a volume of 2·45 m³ with 50·96 days HRT of the Action for Food Production (AFPRO) design. All the constructional items used in the modified plant were in accordance with the recommendations of AFPRO except the number of bricks, i.e. 625 in the modified design as against 800 in the AFPRO design. The rates of biogas production in October 1993 and January 1994 were 0·0357 and 0·0282 m³/kg feed (wet weight of dung); respectively. Thus the modified plant had a gas production efficiency of 70·5–89·4% of its rated capacity. All other functional parameters were within the optimum limits recommended for successful operation of any anaerobic digester.     

Rate constants and activation parameters for organo-cobalt porphyrin bond homolysis from NMR relaxation times

¹H NMR line broadening is found to be an effective complimentary method to chemical trapping for determining the rates and activation parameters for organo-metal bond homolysis events that produce freely diffusing radicals. Application of this method is illustrated by measurement of bond homolysis activation parameters for a series of organo-cobalt porphyrin complexes ((TPP)Co-C(CH₃)₂CN (ΔH^≠ = 19.5±0.9 kcal mol⁻¹, ΔS^≠ = 12±3 cal°K⁻¹ mol⁻¹), (TMP)Co-C(CH₃)₂CN (ΔH^≠ = 20±1 kcal mol⁻¹,ΔS^≠ = 13±2 cal°K⁻¹ mol⁻¹), (TAP)Co-C(CH₃)₂CO₂CH₃ (ΔH^≠ = 18.2±0.5 kcal mol⁻¹, ΔS^≠ = 12±2 cal °K⁻¹ mol⁻¹), (TAP)Co-CH(CH₃)C₆H₅ (ΔH^≠ = 22.5±0.5, ΔS^≠ = 17±2 cal °K⁻¹ mol⁻¹)). The line broadening method is particularly useful in determining activation parameters for dissociation of weakly bonded organometallics where the rate of homolysis can exceed the range measurable by conventional chemical trapping methods.     

Determination of kinetic constants for a two-stage anaerobic upflow packed-bed reactor for dairy wastewater

A two-stage upflow packed-bed (reactors in series) system was used for the treatment of dairy wastewater. Nylon pads were used as supporting media for the biomass. This investigation aimed at the determination of various kinetic constants for substrate, biomass and biogas based on various models. The maximum loadings that could be applied to reactor I and reactor II were 14·29 and 5·0 kg of chemical oxygen demand (COD) per m³ per day, respectively. The maximum COD removal efficiencies at various loading rates were in the ranges of 93·8–98·5% and 72·5–84% for the two reactor systems, respectively. The combined biogas yield was between 0·196 and 0·386 m³ gas/kg COD_a.     

The reaction between the superoxide anion radical and cytochrome c

1. 1. The superoxide anion radical (O₂⁻) reacts with ferricytochrome c to form ferrocytochrome c. No intermediate complexes are observable. No reaction could be detected between O₂⁻ and ferrocytochrome c.

2. 2. At 20 °C the rate constant for the reaction at pH 4.7 to 6.7 is 1.4 · 10⁶ M⁻¹ · s⁻¹ and as the pH increases above 6.7 the rate constant steadily decreases. The dependence on pH is the same for tuna heart and horse heart cytochrome c. No reaction could be demonstrated between O₂⁻ and the form of cytochrome c which exists above pH ≈ 9.2. The dependence of the rate constant on pH can be explained if cytochrome c has pKs of 7.45 and 9.2, and O₂⁻ reacts with the form present below pH 7.45 with k = 1.4 · 10⁶ M⁻¹ · s⁻¹, the form above pH 7.45 with k = 3.0 · 10⁵ M⁻¹ · s⁻¹, and the form present above pH 9.2 with k = 0.

3. 3. The reaction has an activation energy of 20 kJ mol⁻¹ and an enthalpy of activation at 25 °C of 18 kJ mol⁻¹ both above and below pH 7.45. It is suggested that O₂⁻ may reduce cytochrome c through a track composed of aromatic amino acids, and that little protein rearrangement is required for the formation of the activated complex.

4. 4. No reduction of ferricytochrome c by HO₂ radicals could be demonstrated at pH 1.2–6.2 but at pH 5.3, HO₂ radicals oxidize ferrocytochrome c with a rate constant of about 5 · 10⁵–5 · 10⁶ M⁻¹ · s⁻¹

.     

Photosynthetic response of Myriophyllum salsugineum A.E. orchard to photon irradiance, temperature and external free CO2

The photosynthetic capacity of Myriophyllum salsugineum A.E. Orchard was measured, using plants collected from Lake Wendouree, Ballarat, Victoria and grown subsequently in a glasshouse pond at Griffith, New South Wales. At pH 7.00, under conditions of constant total alkalinity of 1.0 meq dm⁻³ and saturating photon irradiance, the temperature optimum was found to be 30–35°C with rates of 140 μmol mg⁻¹ chlorophyll a h⁻¹ for oxygen production and 149 μmol mg⁻¹ chlorophyll a h⁻¹ for consumption of CO₂. These rates are generally higher than those measured by other workers for the noxious Eurasian water milfoil, Myriophyllum spicatum L., of which Myriophyllum salsugineum is a close relative. The light-compensation point and the photon irradiance required to saturate photosynthetic oxygen production were exponentially dependent on water temperature. Over the temperature range 15–35°C the light-compensation point increased from 2.4 to 16.9 μmol (PAR) m⁻² s⁻¹ for oxygen production while saturation photon irradiance increased from 41.5 to 138 μmol (PAR) m⁻² s⁻¹ for oxygen production and from 42.0 to 174 μmol (PAR) m⁻² s⁻¹ for CO₂ consumption. Respiration rates increased from 27.1 to 112.3 μmol (oxygen consumed) g⁻¹ dry weight h⁻¹ as temperature was increased from 15 to 35°C. The optimum temperature for productivity is 30°C.     

Growth and biochemical characterization of microalgal biomass produced in bubble column and airlift photobioreactors: studies in fed-batch culture

Relatively large (0.19 m column diameter, 2 m tall, 0.06 m³ working volume) outdoor bubble column and airlift bioreactors (a split-cylinder and a draft-tube airlift device) were compared for monoseptic fed-batch culture of the microalga Phaeodactylum tricornutum. The three photobioreactors produced similar biomass versus time profiles and final biomass concentration (4 kg m⁻³). The maximum specific growth rate observed within a daily illuminated period in the exponential growth phase, had a value of 0.08 h⁻¹ on the third day of culture. Because of night-time losses of biomass, the specific growth rate averaged over the 4-days of exponential phase was 0.021 h⁻¹ for the three reactors.

The biomass in the vertical column reactors did not experience photoinhibition under conditions (photosynthetically active daily averaged irradiance value of 1150±52 μE m⁻² s⁻¹) that are known to cause photoinhibition in conventional thin-tube horizontal loop reactors. Because of good gas-liquid mass transfer, the dissolved oxygen concentration in the reactors at peak photosynthesis remained <120% of air saturation; thus, oxygen inhibition of photosynthesis and photo-oxidation of the biomass did not occur. Carbohydrate accumulation (up to 13% w/w) by the biomass was favored during light-limited linear growth. A declining light intensity caused a more than five-fold increase in cellular carotenoids but the chlorophylls increased only by about 2.5-fold during the course of the culture. In the stationary phase, up to 2% of the biomass was chlorophylls and carotenoids constituted up to 0.5% of the biomass dry weight.     

Production d'NH4 par la crevette Crangon crangon L. dans deux écosystémes côtiers. approche expérimentale et étude de l'influence du sédiment sur le taux d'excrétion

Estimation of the ammonia production of the shrimp C. crangon in two littoral ecosystems (oligotrophic sand and eutrophic mud) was determined in winter and summer conditions from laboratory observations in experimental microcosms. The ammonia excretion rate of C. crangon was not influenced by either the sediment type or the ammonia concentration of the overlying water; on the other hand, the mean excretion rate and the response to initial handling stress increased markedly as shrimp were deprived of soft substratum.

The daily ammonia production of C. crangon was 16 μmol NH₃ · g ⁻¹ wet wt · day ⁻¹ in winter and 40 μmol in summer. A gross production of 12 μmol NH₃ · m⁻² · day ⁻¹ and 300–700 μmol μ m⁻² · day⁻¹, respectively, could be expected in the two ecosystems studied. This would account for 5% (winter) and 2–4% (summer) of the total NH⁺₄ flux at the sediment-water interface. The contribution of the excretion of all macrofauna to the NH⁺₄ flux from the sediment is discussed.     

Biogas-plant effluent as an organic fertiliser in fish polyculture

Biogas-plant effluent collected from a KVIC model biogas-plant fed on cattle waste was utilised in fish polyculture. Biogas-plant effluent was applied at 0·15% concentration at 3-day intervals. The growth rate of Labeo rohita was 4·52 ±0 ·75 g fish⁻¹ day⁻¹, of Cirrhina mrigala 3·36 ± 0·48 g fish⁻ day⁻¹ and of Cyprinus carpio was 1·82 ± 0·41 g fish⁻¹ day⁻¹. Total fish production was 13·44 ± 0·77 kg 0·002 ha⁻¹ year⁻¹ (6653 kg ha⁻¹ year⁻¹) without any supplementary fish-feed.     

Hydrodynamic properties of the fractions of mannan formed by Rhodotorula rubra yeast

Aqueous solutions of fractions of an extracellular linear mannan formed by Rhodotorula rubra yeast have been investigated by hydrodynamic methods (high-speed sedimentation, translation isothermic diffusion and viscometry). The molecular weight was determined according to Svedberg ( ) and the polydispersity parameters of the initial sample were also determined (M_w/M_n = 1·20 and M_z/M_w = 1·21). Relationships between the molecular weight (M) and s_o, D_o and [η] in the range were: [η] = 2·33 × 10⁻² M^0.75, D_o = 1·65 × 10⁻⁴ M^0·58, s_o = 2·24 × 10⁻¹⁵ M^0·43. The equilibrium rigidity and hydrodynamic diameter of chains representing mannan molecules were evaluated.     

Ricochet inter-ring haptotropic rearrangement of σ-methyl-(η-indenyl) chromium tricarbonyls. Experimental kinetic and theoretical DFT study

σ-Methyl-(η⁵-indenyl) chromium tricarbonyl (III) rearranges quantitatively into η⁶-1-endo-methylindene) chromium tricarbonyl (IV) in C₆D₆ solution at 30–60°C. Methyl group attachment to the positions 2 or 3 of indenyl ligand in (III) has no influence on the activation parameters of this ricochet inter-ring haptotropic rearrangement (ΔG^#=23.6 kcal mol⁻¹; ΔH^#=18.9±0.2 kcal mol⁻¹; ΔS^#=−18.6±0.2 cal K⁻¹ mol⁻¹). (IV) undergoes further irreversible isomerization at 60–120° into (ν⁶-3-methylindene) chromium tricarbonyl (V) with a higher activation barrier (ΔG^#=28.5±0.1 kcal mol⁻¹) via two consecutive [1,5]-sigmatropic hydrogen shifts. The mechanisms of both rearrangements have been studied in detail using density functional theory (DFT) calculations with extended basis sets. Calculations show that the rearrangement (III) → (IV) proceeds in two steps. Methyl group migration from chromium into position 1 of the indenyl ligand is the rate-determining step leading to the formation of the 16-electron intermediate (VII). The calculated activation barrier (E_a=19.6 kcal mol⁻¹) is in good agreement with the experimental one. Further rearrangement (VII) → (V) proceeds via a trimethylenemethane-type transition state (XVIII) with an activation barrier 11.8 kcal mol⁻¹. The coordination of the chromium tricarbonyl group at the six-membered ring has only minor influence on the kinetic parameters of the hydrogen [1,5]-sigmatropic shift in indene.