A novel multienzyme electrode for the determination of citrate

A novel amperometric biosensor for the determination of citric acid in food samples and fermentation broths has been developed. The sensor is composed of citrate lyase (CL, EC 4.1.3.6), oxaloacetate decarboxylase (OAC, EC 4.1.1.3) and pyruvate oxidase (POP, EC 1.2.3.3), co-immobilized in gelatin, and an amperometric transducer. A Clark-type O₂-electrode and a modified Clark-type H₂O₂-electrode were alternatively used as a transducer. The biosensor covers a linear detection range from 1 μmol dm^?3 to 1 mmol dm^?3 citrate, with a response time of 2·5 min for the steady state response. The lower detection limit for citrate is 0·5 μmol dm^?3. The response of the sensor remained constant for 8 days and decreased to 25% after 18 days at 20–23°C. The results obtained from citrate determinations in food samples and fermentation broths agree well with those determined by enzymatic sample anlaysis. The relative standard deviation for citrate determinations with the new biosensor was 2·2% (n = 7).     

Biocatalysis of Chlorophyllase in Ternary Micellar Systems Using Pheophytins as Substrates

The partially purified chlorophyllase, obtained from the alga Phaeodactylum tricornutum, was assayed for its hydrolytic activity towards the pheophytin in ternary micellar systems of hexane/Tris–HCl/surfactant. A wide range of surfactants, sorbitans (Span 20, 40, 60, 80 and 85) and polysorbates (Tween 20, 40, 60, 80 and 85), was used. The use of either 50 μmol dm⁻³ of Span 85 or 1 μmol dm⁻³ of Tween 80 increased the hydrolytic activity of chlorophyllase by 110 and 23%, respectively. The optimum values of pH, enzyme content, incubation time and temperature for the hydrolytic activity of chlorophyllase were determined as 8·25, 8·00 μg protein cm⁻³, 60 min and 27·5°C, respectively. The V_max and K_m values were 6·91 nmole hydrolyzed pheophytin mg⁻¹ protein min⁻¹ and 47·2 nmole pheophytin dm⁻³, respectively, in the Span 85 medium and 10·04 and 121·00, respectively, in the Tween 80 medium. The addition of optimized amounts of individual membrane lipids, L -α-phosphatidylcholine, L -α-phosphatidyl-DL -glycerol and β-carotene increased the hydrolytic activity of chlorophyllase by 50, 36 and 10%, respectively, for Span 85 and 30, 48 and 15%, respectively, for Tween 80. Phytol showed a competitive inhibitory effect on chlorophyllase activity in both Span and Tween systems with a K_i value of 15·5 and 14·3 μmol dm⁻³, respectively. High-performance liquid chromatography and spectrophotometry analyses were used to characterize the end-products of chlorophyllase hydrolytic reaction. © 1997 SCI.     

Production of optically pure L-alanine by immobilized Pseudomonas sp. BA2 cells

The conditions for immobilizing the new L -aminoacylase-producing bacterial strain, Pseudomonas sp. BA2, by entrapment in κ-carrageenan gel, were investigated. The optimal gel concentration and cell load were determined. The addition of CoCl₂ and N-acetyl-L -alanine to the immobilizing matrix enhanced L -aminoacylase activity. The enzymatic properties of immobilized Pseudomonas sp. BA2 were investigated. Enzyme activity in immobilized cells was optimal at a pH of 6·5 using 0·15 mol dm⁻³ Tris–maleate buffer at 45°C. The presence of 0·7 mmol dm⁻³ CoCl₂ in the enzymatic reaction mixture improved L -aminoacylase activity. The immobilized cell preparation was used for the production of L -alanine from N-acetyl-DL -alanine in a batch reactor. Conversions of 100% were obtained using substrate concentrations ranging from 20 to 200 mmol dm⁻³. The reactor production was 0·74 mol h⁻¹ g cell⁻¹ dm⁻³ which is noticeably higher than that previously reported in the literature. © 1998 Society of Chemical Industry     

Factors affecting silver biosorption by an industrial strain of Saccharomyces cerevisiae

Factors affecting silver biosorption by Saccharomyces cerevisiae biomass, obtained as a waste product from industry, were examined. Maximum removal of silver from solution was achieved within 5 min. Increasing the concentration of biomass in experimental flasks from 1 to 8 mg cm⁻³ decreased both silver accumulation, from 224·7 to 89·5 μmol Ag g⁻¹ dry wt, and associated H⁺ ion release, from 109·4 to 31·7 μmol H⁺ g⁻¹ dry wt. The presence of 1·0 mol dm⁻³ cadmium or methionine decreased silver biosorption by 40% and 93% respectively. Boiling in 100 mmol dm⁻³ NaOH or 10 mmol dm⁻³ sodium dodecyl sulphate decreased silver biosorption by 54% and 25% respectively. A temperature increase from 4°C to 55°C decreased silver biosorption by 9%. The metabolic state of the yeast had no effect on silver biosorption. Decreasing the pH of the silver solution caused a reduction in metal removal by the biomass.     

Photocatalytic degradation of diuron [3-(3,4-dichlorophenyl)-1,1-dimethylurea] on the layer of TiO2 particles in the batch mode plate film reactor

The photocatalytic degradation of diuron in aqueous solution on TiO₂ layer of a batch mode plate reactor irradiated with ultraviolet sun-bed tubes was investigated. Dependence of the reaction rate on the diuron concentration (in the range of 0·8–8·0×10⁻⁵ mol dm⁻³) and on the light intensity (0·8–2·7×10⁻⁹ einstein cm⁻² s⁻¹) but independence on the flow rate (2·5–3·6 dm³ min⁻¹) were found. A reaction scheme was proposed following the main identified primary [3-(3,4-dichlorophenyl)-1-methyl-1-formylurea and 3-(3,4-dichlorophenyl)-1-methylurea] and secondary [3-(3,4-dichlorophenyl)-1-formylurea and 3,4-dichlorophenylurea] products. © 1998 SCI     

Removal of Hydrogen Sulphide by Immobilized Thiobacillus sp. strain CH11 in a Biofilter

An autotropic Thiobacillus sp. CH11 was isolated from piggery wastewater containing hydrogen sulphide. The removal characteristics of hydrogen sulphide by Thiobacillus sp. CH11 were examined in the continuous system. The hydrogen sulphide removal capacity was elevated by the BDST (Bed Depth Service Time) method (physical adsorption) and an immobilized cell biofilter (biological conversion). The optimum pH to remove hydrogen sulphide ranged from 6 to 8. The average specific uptake rate of hydrogen sulphide was as 1·02×10⁻¹³ mol-S cell⁻¹ h⁻¹ in continuous systems. The maximum removal rate and saturation constant for hydrogen sulphide were calculated to be V_m = 30·1 mmol-S day⁻¹ (kg-dry bead)⁻¹ and K_s = 1·28 μmol dm⁻³, respectively. A criterion to design a scale-up biofilter was also studied. The maximum inlet loading in the linear region (95% removal) was 47 mmol-S day⁻¹ (kg-dry bead)⁻¹. Additionally, the biofilter exhibited high efficiency (>98·5%) in the removal of hydrogen sulphide at both low (<0·026 mg dm⁻³) and high (0·078 mg dm⁻³) concentrations. The results suggested that the Thiobacillus sp. CH11 immobilized with Ca-alginate is a potential method for the removal of hydrogen sulphide. © 1997 SCI.     

Electroplating of cobalt from aqueous citrate baths

Electroplating of cobalt onto steel substrates from citrate baths has been investigated under different conditions of bath composition, current density, pH and temperature. A detailed study has been made of the influence of these variables on the potentiodynamic cathodic polarization curves, cathodic current efficiency and the throwing power as well as the throwing index of these baths. The optimum bath composition has been established and it contains: CoSO₄.7H₂O (0·36 mol dm⁻³) trisodium citrate (0·19 mol dm⁻³) and citric acid (0·1 mol dm⁻³) at pH 5·0. The microhardness of cobalt electrodeposited from citrate baths is high and it may be, under certain conditions, two or three times higher than that reported for cobalt electrodeposited from other different baths. The surface morphology of the as-plated cobalt was investigated by using scanning electron microscopy (SEM) while the structure was studied by using X-ray diffraction analysis and anodic stripping voltammetry (ASV) techniques. © 1998 Society of Chemical Industry     

Tris (2,2′-bipyridil) copper (II) chloride complex: a biomimetic tyrosinase catalyst in the amperometric sensor construction

The use of tris (2,2′-bipyridil) copper (II) chloride complex, [Cu(bipy)₃]Cl₂·6H₂O, as a biomimetic catalyst, is reported in the construction of an amperometric sensor for dopamine. The sensor was prepared modifying a glassy carbon electrode with a Nafion® membrane doped with the complex. The optimized conditions for the sensor response were obtained in 0.25 mol dm⁻³ Pipes buffer (pH 7.0) containing 150 μmol dm⁻³ H₂O₂, with an applied potential of −50 mV versus saturated calomel electrode (SCE). In these conditions, a linear response range between 9 and 230 μmol dm⁻³, with a sensitivity of 1.43±0.01 nA dm³ μmol⁻¹ cm⁻² and a detection limit of 4.8 μmol dm⁻³ were observed for dopamine. The response time for this sensor was about 1 s, presenting the same response for at least 150 successive measurements, with a good repeatability (4.8%) expressed as relative standard deviation for n=13. After its construction, this sensor can be used after 180 days without loss of sensitivity, kept at room temperature. The difference of the sensor response between four preparations was 4.2%. A detailed investigation about the sensor response for other eighteen phenolic compounds and five interfering species was performed. The sensor was applied for dopamine determination in pharmaceutical preparation with success.     

Co-digestion of Municipal Sewage Sludges and Pre-hydrolysed Woody Agricultural Wastes

Material balance has been used to evaluate the COD behaviour and the time required for fed-batch digestion of mixtures of domestic sludges and pre-hydrolysed agricultural wastes. Pre-hydrolysis of the feed materials has been used to penetrate the strong lignocellulosic structure of these wastes as well as to increase the fraction of soluble organic substances in the mixture. The influence of the organic loading rate on the main process parameters (methane, carbon dioxide, total biogas productions and their respective conversion yields) has also been investigated. The organic load has been varied from 0·8 up to 6·1 g_COD dm⁻³ day⁻¹, corresponding to a range of volatile solids load of 0·6–4·5 g_VS dm⁻³ day⁻¹ for the material under consideration. These values are slightly higher than those usually employed in conventional digester for domestic sewage sludges. However, methane production reached a maximum rate of only 5·6 mmol dm⁻³ day⁻¹ at an organic loading rate of 4·6 g_COD dm⁻³ day⁻¹, while both CH₄ content and production of biogas rapidly fell over 2·2 g_COD dm⁻³ day⁻¹. On the whole, these results suggest that removal of lignin is necessary in order to carry out the continuous anaerobic digestion of pre-hydrolysed agricultural wastes rich in woody materials. © 1997 SCI.     

Influence of growth medium composition on the lipolytic enzyme activity of Ophiostoma piliferum (CartapipTM)

Production of esterases (carboxyl esterase EC 3.1.1.1) and lipases (glycerol ester hydrolase EC 3.1.1.3) by Ophiostoma piliferum (Cartapip^TM), a fungus commercialized to decrease fatty acyl esters in wood, is described. The influence of various combinations of carbon and nitrogen sources, in the growth medium, was examined. Medium containing yeast extract as a nitrogen source and olive oil as a carbon source was found to be optimal for extracellular esterase (221 U dm⁻³) and lipase (152 U dm ⁻³) activities. Further increases in those enzyme activities were achieved by decreasing medium pH from 6.5 to 5.5 (esterase 508 U dm⁻³; lipase 415 U dm ⁻³) and increasing medium calcium content from 8 m mol dm⁻³ to 160 m mol dm⁻³ (esterase 4084 U dm⁻³; lipase 508 U dm ⁻³) © 1999 Society of Chemical Industry     

Technological and kinetic aspects of sublethal acid toxicity in microbial gum production

Chemostat culture of Xanthomonas campestris were obtained at a dilution rate of 0·05 h⁻¹ and the normal feed then supplemented with 0·58 and 1·74 mmol dm⁻³ isobutyric acid (IBA). Data revealed that the organism responded to sublethal acid stress by overproducing xanthan. The acid additions led to transient zones in the continuous cultivation profiles. By adding feed containing 1·74 mmol dm⁻³ IBA, volumetric growth rate immediately decreased from 0·059 to 0·026 g dm⁻³ h⁻¹ whereas the specific xanthan formation rate increased from 0·23 g g⁻¹ biomass h⁻¹ to a maximum 0·65 g g⁻¹ biomass h⁻¹ (with 1·0 mmol dm⁻³ IBA addition), before decreasing as the concentration of acid attained that of the feed. By monitoring the outlet CO₂ in parallel with biomass and polysaccharide levels in the IBA fermentation a 10% diversion of the total carbon flux from biomass synthesis to xanthan biosynthesis was detected. A consistent pattern of variation in activity was detected in enzymes of intermediary metabolism, suggesting an action at the regulatory level. Enhanced activities of carbon catabolism and xanthan anabolic reactions (phosphomannose isomerase) were observed in the presence of the acid. Batch experiments carried out in the pres-ence of IBA gave results which correlated with the undissociated acid form con-centration. An undissociated acid fraction of 6·5×10⁻³ mmol dm⁻³ was calculated in a set of flasks under the same conditions and a statistically vali-dated 12% increase in xanthan production was found. The maximum activation was determined to be below 1·1×10⁻² mmol dm⁻³ when a 58% specific xanthan production rate increase occurred in parallel with a 35% decrease in biomass concentration.     

Adsorption of cadmium and zinc from aqueous solution on natural and activated bentonite

The adsorption of cadmium and zinc ions on natural bentonite heat-treated at 110°C or at 200°C and on bentonite acid-treated with H₂SO₄ (concentrations: 0·5 mol dm^?3 and 2·5 mol dm^?3), from aqueous solution at 30°C has been studied. The adsorption isotherms corresponding to cadmium and zinc may be classified respectively as H and L types of the Giles classification which suggests the samples have respectively a high and a medium affinity for cadmium and zinc ions. The experimental data points have been fitted to the Langmuir equation in order to calcualte the adsorption capacities (X_m) and the apparent equilibrium constants (K_a) of the samples; X_m and K_a values range respectively for 4·11 mg g^?1 and 1·90 dm³ g^?1 for the sample acid-treated with 2·5 mol dm^?3 H₂SO₄ [(B)-A(2·5)] up to 16·50 mg g^?1 and 30·67 dm³ g^?1 for the natural sample heat-treated at 200°C [B-N-200], for the adsorption process of cadmium, and from 2·39 mg g^?1 and 0·07 dm³ g^?1, also for B-A(2·5), up to 4·54 mg g^?1 and 0·45 dm³ g^?1 [B-N-200], for the adsorption process of zinc. X_m and K_a values for the heat-treated natural samples were higher than those corresponding to the acid-treated ones. The removal efficiency (R) has also been calculated for every sample; R values ranging respectively from 65·9% and 8·2% [B-A(2·5)] up to 100% and 19·9% [B-N-200], for adsorption of cadmium and zinc.     

The influence of ammonium and methods for removal during the anaerobic treatment of poultry manure

The addition of exogenous NH₄Cl to poultry manure and synthetic medium was used to study the effect of ammonia-nitrogen on the activity and composition of a methanogenic consortium. Results indicated that the production of biogas and methane was not affected by the variation in NH₄Cl concentration within the range 2–10 g dm⁻³ (0·5–2·6 g N-NH₄ dm⁻³). At higher values of ammonium (10–30 g dm⁻³ or 2–8 g N-NH₄ dm⁻³) a significant decline in both parameters (by 50–60% for biogas and 80–90% for methane) was observed. A significant decrease in the numbers of bacteria of all physiological groups (especially proteolytic and methanogenic) was observed when more than 30 g NH₄Cl dm⁻³ (7·8 g N-NH₄ dm⁻³) was added to the fermentation medium. The addition of 10% (w/v) of powdered phosphorite ore enhanced the production of biogas and methane at NH₄Cl concentrations up to 30 g dm⁻³, and also changed the composition of the methanogenic consortium. A partial recovery in the numbers of proteolytic and methanogenic bacteria coupled with the decrease in the density of sulphate-reducers was observed. High concentrations (more than 50 g dm⁻³) of NH₄Cl seemed to cause irreversible inhibition of methanogenesis which could not be eliminated by the addition of phosphorites. ©1997 SCI     

Novel porous matrix and bioreactors for high density cultures of insulinoma cell lines: Insulin secretion and response to glucose

This paper describes the use of a novel porous matrix, Porocell, for high density, tissue-like culture of two insulinoma cell lines, CRI-D2 and CRI-D11. Both these cell lines have previously been shown not to secrete insulin in response to glucose. Porocell is a macro-porous, polymeric material manufactured in the shape of discs that are 6·2 mm in diameter and 2 mm in thickness. Insulinoma cells were cultured in two different mini-bioreactors, each containing six Porocell discs inoculated with 2·5 × 10⁶ cells per disc. In surface aerated, stirred bioreactors, the insulinoma cells grew as closely packed dense cell sheets penetrating deep into the pores of Porocell. In a second type of system, a packed-bed perfused mini-bioreactor, flat, extended monolayers of cells were observed growing throughout the Porocell matrix. In both bioreactor configurations, viable cell populations were maintained for 30 days because of the excellent oxygen and nutrient transfer properties of Porocell. CRI-D2 insulinoma cells cultured in static flasks and on Porocell did not show any insulin secretion in response to 30 min exposures in media supplemented with 5·5–16·7 mmol dm⁻³ glucose. However, in long term (14–19 day) cultures, CRI-D2 cells growing in Porocell secreted low, but measurable amounts (25–35 pmol dm⁻³) of insulin in medium supplemented with elevated (14·5 mmol dm⁻³) glucose concentrations. The glucose uptake rates of cells cultured in 4·0 mmol dm⁻³ glucose increased linearly from 1·0 to 2·3 mmol dm⁻³ day⁻¹ over a period of 19 days. At 14·5 mmol dm⁻³ glucose concentration, the uptake rate increased from 1·0 to 7·05 mmol dm⁻³ day⁻¹ over the same period of culture. Contrary to previous studies, we have demonstrated that the CRI-D2 cell line cultured at high cell density in Porocell is capable of secreting insulin when exposed to prolonged and elevated concentrations of glucose. The Porocell mini-bioreactors are easy to use, robust systems that can be used for long-term studies of primary and tumorgenic islet cell function and response to secretagogues. © 1998 SCI.     

Fourier-transform infrared assay of bile salt-stimulated lipase activity in reversed micelles

A Fourier-transform infrared (FT-IR) spectroscopic method has been developed for assaying the bile salt-stimulated human milk lipase (BSSL, EC3.1) catalyzed hydrolysis of triolein in AOT reversed micelles in iso-octane. At 37°C in 50 mmol dm^?3 AOT the molar absorbtivities for the carbonyl stretching frequencies for triolein (at 1751 cm^?1) and oleic acid (at 1714 cm^?1) were 1646 dm³ mol^?1 cm^?1 and 743 dm^?3 mol^?1 cm^?1, respectively. The rate was linearly dependent upon the concentration of enzyme in the water pool up to 10 mg cm^?3 and maximum activity was observed at a ratio (w₀) of [H₂O]:[AOT] = 16·7. Using these conditions, and in the presence of 10 mmol dm^?3 sodium taurocholate (TC), the derived Michaelis–Menten parameters V_max and K_m were 57·5 μmol min^?1 mg^?1 and 5·53 mmol dm^?3, respectively. These results are compared with those obtained in an oil-in-water microemulsion system and are discussed in terms of the relative partitioning of the enzyme and the substrate in the aqueous and oil phases and the interfacial concentration of the substrate in the two systems.     

Adsorption of chlorophyll-a from acetone solution on natural and activated bentonite

The adsorption of chlorophyll-a on bentonite desiccated at 110°C, untreated and acid-treated with H₂SO₄ solutions over a concentration range between 0·25 and 2·50 mol dm^?3, from acetone solution at 25°C has been studied. The adsorption isotherms may be classified as using Giles' classification, as type S (untreated sample and 0·25 mol dm^?3 H₂SO₄-treated sample), type H (0·50 mol dm^?3 H₂SO₄-treated sample) and type L (1·00 and 2·50 mol dm^?3 H₂SO₄-treated samples). This fact suggests that the bentonite surfaces (low, high and medium affinity, respectively) behave in differently relation to the adsorption of the chlorophyll-a molecules. The experimental data points have been fitted to the Freundlich equation in order to calculate the adsorption capacities (K_f) of the samples; K_f values range from 0·43 mg kg^?1 for the untreated bentonite up to 108·89 mg kg^?1 for the 0·50 M H₂SO₄-treated bentonite. The removal efficiencies (R) have also been calculated and range from 5·71% for the untreated bentonite up to 85·18% for the 0·50 M H₂SO₄-treated bentonite.     

Removal of 1,1′-Dimethyl-4,4′bipyridyl Dichloride from Aqueous Solution by Natural and Activated Bentonite

The sorption of 1,1′-dimethyl-4,4′bipyridilium dichloride (paraquat) on bentonite desiccated at 110°C untreated, and acid-treated with H₂SO₄ solutions over a concentration range between 0·25 M and 1·00 M , from aqueous solution at 30°C has been studied by using batch experiments. In addition, column experiments were carried out with the bentonite sample treated with the 1·00 M H₂SO₄ solution [B-A(1·00)] by using two aqueous solutions of paraquat of different concentrations (C = 29·40 mg dm⁻³ and C = 65·38 mg dm⁻³). The experimental data points have been fitted to the Langmuir equation in order to calculate the sorption capacities (X_m) of the samples; X_m values range from 1·35×10⁵ mg kg⁻¹ for the sample acid-treated with 0·375 M H₂SO₄ [B-A(0·375)] up to 1·96×10⁵ mg kg⁻¹ for the untreated bentonite [B-N]. The removal efficiency (R) has also been calculated; R values ranging from 44·61% for the [B-A(0·375)] sample up to 67·23% for B-N. The batch experiments show that the natural bentonite is more effective than the acid-treated bentonite in relation to sorption of paraquat. The column experiments show that the B-A(1·00) sample might be reasonably used in removing paraquat, the column efficiency increasing from 37·55% for the C = 65·38 mg dm⁻³ aqueous solution of paraquat up to 66·58% for the C = 29·40 mg dm⁻³ one. © 1997 SCI.     

Characterization of composite membrane of poly(vinyl alcohol) and regenerated silk fibroin for immobilization of horseradish peroxidase and an amperometric neckelocene-mediated sensor sensitive to hydrogen peroxide

Horseradish peroxidase (HRP) was effectively entrapped in a novel composite membrane of poly(vinyl alcohol) and regenerated silk fibroin. IR spectroscopy was employed to characterize the structure of the composite membrane and scanning electron microscopy was used to visualize the morphology of the composite membrane. HRP was utilized to amplify the amperometric response of the sensor by catalyzing reduction of hydrogen peroxide via nickelocene, an electron transfer mediator between immobilized HRP and a glassy carbon electrode. The influence of various experimental parameters such as pH, temperature and applied potential were explored for optimum analytical performance. The sensor possessed a variety of characteristics including good sensitivity, rapid response time and a low detection of limit of 0·1 μmol dm⁻³, which arose from the efficiency of electron transfer between immobilized HRP and the electrode via nickelocene.     

Nitrification of fertilizer wastewaters in a biofilm reactor

The nitrification characteristics of fertilizer wastes were investigated in a biofilm system using a submerged aerated filter. The attachment of biomass on packing media was studied. Supplement of organic carbon in the form of glucose and yeast extract enhanced biofilm formation although the nitrifiers did not require organic carbon for growth. After an attachment period, continuous operation of the reactor at different loading rates and dissolved oxygen levels was investigated. The maximum achievable nitrification rate was strongly dependent on the dissolved oxygen. In the dissolved oxygen range of 3·2–3·5 mg dm⁻³, the maximum ammonia removal rate was about 0·17 kg NH₄ N m⁻³ day⁻¹. When the dissolved oxygen was increased to 4·9 mg dm⁻³, removal rates as high as 0·41 kg NH₄ N m⁻³ day⁻¹ could be obtained. Nitrite accumulation depended on the bulk nitrogen and dissolved oxygen concentrations.     

Suitability of Sodium Pyruvate as Decomposer of Carried-over Liquid Hydrogen Peroxide

The aim of the present study was to evaluate the concentration of sodium pyruvate in growth media by using the most-probable number method to decompose carried-over liquid hydrogen peroxide (H₂O₂) without negative influences on correct enumeration of Geobacillus stearothermophilus spores. An equivalent molar ratio of sodium pyruvate and H₂O₂ was verified to be sufficient for a complete decomposition. The results showed that by exposition of G. stearothermophilus spores in different liquid hydrogen peroxide and by carry-over of concentrations ranging from 3.56 · 10⁻⁶ up to 1.69 · 10⁻⁴ mol, a sodium pyruvate concentration of 0.05 mol L⁻¹ in growth media was most efficient for the recovery of spores.