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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.151, #2-3)

Effect of Matrix Elasticity on the Continuous Foaming of Food Models by I. Narchi; Ch. Vial; G. Djelveh (pp. 105-121).

The aim is to understand the effect of matrix elasticity on continuous foaming using food models based on glucose syrup. This was modified by adding polyacrylamide (PAA) with 2% whey protein isolate (WPI) or Tween 80 as foaming agents. Foaming was conducted in a stirred column. Rotation speed N and gas-to-liquid flow ratio (G/L) were varied. Overrun, average bubble size d ₃₂, texture and stability were measured using densimetry, image analysis, and rheometry, respectively. Experimental results showed that 0.01% PAA did not modify the viscosity of 2% WPI models, but conferred low elastic behavior. PAA (0.05%) doubled matrix viscosity and drastically increased elasticity. The increase of elasticity became slower for further PAA addition. Foaming experiments demonstrated that theoretical overrun could not be achieved for inelastic WPI models in two cases: for high viscosity and low N, as dispersion effectiveness was reduced; for high G/L and N because of enhanced coalescence. Matrix elasticity was shown to increase overrun at constant viscosity for high G/L by enhancing interface stabilization. However, in elastic models, gas dispersion was more difficult and d ₃₂ was higher than in inelastic fluids of similar viscosity. Finally, when the limiting step was dispersion, foaming was shown to be negatively affected by matrix elasticity.

Keywords: Foamed food; Foaming process; Matrix elasticity; Matrix viscosity

Cellulase Production Under Solid-State Fermentation by Trichoderma reesei RUT C30: Statistical Optimization of Process Parameters by Naveen Kumar Mekala; Reeta Rani Singhania; Rajeev K. Sukumaran; Ashok Pandey (pp. 122-131).

Sugar cane bagasse was used as substrate for cellulase production using Trichoderma reesei RUT C30, and the culture parameters were optimized for enhancing cellulase yield. The culture parameters, such as incubation temperature, duration of incubation, and inducer concentration, were optimized for enhancing cellulase yield using a Box–Behnken experimental design. The optimal level of each parameter for maximum cellulase production by the fungus was determined. Predicted results showed that cellulase production was highest (25.6 FPAase units per gram dry substrate) when the inducer concentration was 0.331 ml/gds, and the incubation temperature and time were 33 °C and 67 h, respectively. Crude inducer generated by cellulase action was found to be very effective in inducing cellulases. Validation of predicted results was done, and the experimental values correlated well with that of the predicted.

Keywords: Cellulase; Trichoderma reesei ; Solid-state fermentation; Sugar cane bagasse; Box–Behnken; Bioethanol

Polycyclic Aromatic Hydrocarbons (PAHs) Biodegradation by Basidiomycetes Fungi, Pseudomonas Isolate, and Their Cocultures: Comparative In Vivo and In Silico Approach by A. Arun; P. Praveen Raja; R. Arthi; M. Ananthi; K. Sathish Kumar; M. Eyini (pp. 132-142).

The polycyclic aromatic hydrocarbons (PAHs) biodegradation potential of the five basidiomycetes’ fungal monocultures and their cocultures was compared with that of a Pseudomonas isolate recovered from oil-spilled soil. As utilization of hydrocarbons by the microorganisms is associated with biosurfactant production, the level of biosurfactant production and its composition by the selected microorganisms was also investigated. The Pseudomonas isolate showed higher ability to degrade three of the five PAHs but the isolate did not produce biosurfactant higher than C. versicolor and P. ostreatus. Among the PAHs, the most effective biodegradation of PAH—pyrene (42%)—was obtained with the fungus C. versicolor. Cocultures involving the fungi and Pseudomonas could not significantly degrade the selected PAHs compounds above that degraded by the most efficient monoculture. A slight increase in pyrene degradation was observed in cocultures of C. versicolor and F. palustris (93.7% pyrene). The crude biosurfactant was biochemically characterized as a multicomponent surfactant consisting of protein and polysaccharides. The PAH biodegradation potential of the basidiomycetes fungi positively correlated with their potential to express ligninolytic enzymes such as lignin peroxidase (Lip), manganese peroxidase (Mnp), and laccase. The present study utilized in silico method such as protein–ligand docking using the FRED in Open Eye software as a tool to assess the level of ligninolytic enzymes and PAHs interactions. The in silico analysis using FRED revealed that of the five PAHs, maximum interaction occurred between pyrene and all the three ligninolytic enzymes. The results of the in silico analysis corroborated with our experimental results showing that pyrene was degraded to the maximum extent by species such as C. versicolor and P. ostreatus.

Keywords: PAHs; Biodegradation; Ligninolytic enzymes; Biosurfactant; In silico method; Docking

Optimization of Crucial Reaction Conditions for the Production of Nicotinamide by Nitrile Hydratase Using Response Surface Methodology by Ashwini Kamble; U. C. Banerjee (pp. 143-150).

The reaction conditions for the nicotinamide production by Rhodococcus erythropolis MTCC 1526 have been optimized by statistical experimental design. Application of this approach in the bioprocess can result in rallied product yield, reduced development time, and process variability. In this investigation, response surface methodology and central composite design were employed to predict the levels of variables such as reaction pH (6.5, 7, and 7.5), temperature (15, 20, and 25 °C), cell concentration (190, 200, and 210 mg/ml), and substrate concentration (18, 20, and 22 mM) on the production of nicotinamide. A total of 22 experiments were carried out in shake flasks, and a three-dimensional response surface was generated to determine the effect of crucial reaction parameters for the maximum conversion of 3-cynopyridine to nicotinamide. Using this methodology, the optimal values for the reaction conditions were reaction pH 6.85, temperature of 24.8 °C, cell concentration of 190.98 mg/ml, and substrate concentration of 21.98 mM. This statistical approach led to the increase of conversion of 3-cynopyridine (93%) as compared to the conversion obtained by one-factor-at-a-time approach (84%).

Keywords: Nitrile hydratase; Nicotinamide; 3-cyanopyridine; Response surface methodology; Reaction optimization; Reaction temperature

Evaluation of Lignocellulosic Wastes for Production of Edible Mushrooms by P. Rani; N. Kalyani; K. Prathiba (pp. 151-159).

The degradation of lignocellulosic wastes such as paddy straw, sorghum stalk, and banana pseudostem was investigated during solid-state fermentation by edible mushrooms Pleurotus eous and Lentinus connotus. Biological efficiency of 55–65% was observed in paddy straw followed by sorghum stalk (45%) and banana pseudostem (33%) for both fungal species. The activity of extracellular enzymes, namely cellulase, polyphenol oxidase, and laccase, together with the content of cellulose, lignin, and phenols, was studied in spent substrates on seventh, 17th, and 27th days of spawning, and these values were used as indicators of the extent of lignocellulosic degradation by mushroom. Both the mushroom species proved to be efficient degraders of lignocellulosic biomass of paddy straw and sorghum stalk, and the extent of cellulose degradation was 63–72% of dry weight (d.w.), and lignin degradation was 23–30% of the d.w. In banana pseudostem, the extent of the degradation was observed to be only 15–22% of the d.w. for both lignin and cellulose. Preferential removal of cellulose during initial growth period and delayed degradation of lignin were observed in all three substrates. This is associated with decrease in activity of cellulase and polyphenol oxidase and increase in laccase activity with spawn aging in spent substrates. Thus, bioconversion of lignocellulosic biomass by P. eous and L. connotus offers a promising way to convert low-quality biomass into an improved human food.

Keywords: Agricultural waste recycling; Edible mushrooms; Lentinus connotus ; Pleurotus eous ; Biological efficiency; Lignocellulolytic activity

Protein-Coated Microcrystals of Pseudomonas aeruginosa PseA lipase by Ruchi Gaur; G. N. Gupta; M. Vamsikrishnan; S. K. Khare (pp. 160-166).

Highly active Pseudomonas aeruginosa lipase protein-coated microcrystals (PAL PCMC) have been prepared by immobilization of protein onto K₂SO₄ as excipient solid support carrier and n-propanol as precipitating solvent. Transmission electron micrographs confirmed the formation of PAL PCMC. These PCMC were found to be a catalytically more active and stable preparation for p-nitrophenyl palmitate hydrolysis in n-heptane, compared to free lipase. The V _max, K _m, and temperature optimum for PAL PCMC increased from 0.49 to 5.66 nmol min⁻¹ mg⁻¹, 589 to 679.8 mmol, and 40°C to 45°C, respectively. These were thermally more stable with 4.65, 2.56, and 1.24-fold improvement in half lives at 45°C, 55°C, and 60°C compared to free P. aeruginosa PseA lipase. Their catalytic efficiency was enhanced by tenfold over that of free enzyme. PAL PCMC offer a simple and effective technique for obtaining stable and efficient lipase preparation for biocatalysis in nonaqueous medium.

Keywords: Pseudomonas aeruginosa ; Protein-coated microcrystals (PCMC); Lipase; Immobilization; Nonaqueous medium

Antioxidant and Hepatoprotective Activity of a Lichen Usnea ghattensis in Vitro by Neeraj Verma; B. C. Behera; Urmila Makhija (pp. 167-181).

Antioxidative and hepatoprotective activity of a cultured lichen Usnea ghattensis has been studied. The methanolic extract of cultured lichen U. ghattensis showed good antioxidant activity by preventing lipid peroxidation by 67% and 86% in Trolox-equivalent antioxidant capacity at 20 μg/ml. It also showed superoxide, 1,1-diphenyl-2-picrylhydrazyl, nitric oxide, and hydroxyl radical-scavenging activity, 89%, 89.6%, 94.8%, and 89.6%, respectively, and found levels higher then that known for the synthetic antioxidants butylated hydroxytoluene, butylated hydroxyanisol, and quercetin at 20 μg/ml concentration. The cultured lichen extract also showed hepatoprotection against ethanol-induced toxicity in the mice liver slice culture model by a significant decrease in the antioxidant enzymes, glutathione peroxidase, catalase, and superoxide dismutase, along with a decrease in lipid peroxidation and lactate dehydrogenase release.

Keywords: Lichen; Usnea ghattensis ; Antioxidant activity; Hepatoprotective activity

Application of Response Surface Methodology for Maximizing Dextransucrase Production from Leuconostoc mesenteroides NRRL B-640 in a Bioreactor by Ravi Kiran Purama; Arun Goyal (pp. 182-192).

The production of dextransucrase from Leuconostoc mesenteroides NRRL B-640 was investigated using statistical approaches. Plackett–Burman design with six variables, viz. sucrose, yeast extract, K₂HPO₄, peptone, beef extract, and Tween 80, was used to screen the nutrients that significantly affected the dextransucrase production. 2⁴-Central composite design with four selected variables (sucrose, K₂HPO₄, yeast extract, and beef extract) was used for response surface methodology (RSM) for optimizing the enzyme production. The culture was grown under flask culture with 100 ml optimized medium containing 30 g/l sucrose, 18.5 g/l yeast extract, 15.3 g/l K₂HPO₄, and 5 g/l beef extract at 25 °C and shaking at 200 rpm gave dextransucrase with specific activity of 0.68 U/mg. Whereas the same optimized medium in a 3.0-l bioreactor (1.4 l working volume) gave an experimentally determined value of specific activity of 0.70 U/mg, which was in perfect agreement with the predicted value of 0.65 U/mg by the statistical model.

Keywords: Dextransucrase; Leuconostoc mesenteroides ; RSM; Plackett–Burman; Central composite design

Biotreatment of High Strength Nitrate Waste Using Immobilized Preadapted Sludge by Rashmi R. Nair; Pradip B. Dhamole; S. S. Lele; Stanislaus F. D’Souza (pp. 193-200).

One of the major wastes generated by fertilizer, explosive, and nuclear industries are nitrate (as high as 1,000 ppm NO₃N) whose removal before disposal has become a growing concern. In this study, an active denitrifying sludge was immobilized onto support materials like cloth and polyurethane foam and their denitrification efficiency on high nitrate wastes [1,000 ppm NO₃ (225 ppm NO₃N), 5,000 ppm NO₃ (1,129 ppm NO₃N), 7,500 ppm NO₃ (1,693 ppm NO₃ N)] was studied. Results showed complete degradation of the nitrate wastes (225 ppm NO₃N, 1,129 ppm NO₃N, and 1,693 ppm NO₃N) without any accumulation of nitrite in a period of only 1, 4, and 10 h, respectively. Based on adhering and entrapment principle, an immobilization unit was developed using a combination of cloth and foam as well as both individually. This system used for treating such high nitrate wastes was found to be quite effective in waste water treatment, particularly in problems associated with solid–liquid separation. The batch column reactor was run in about 45 batches without any loss in activity or reactor stability.

Keywords: Denitrification; Immobilization; Cloth; Polyurethane foam; Activated sludge

Application of a Data Reconciliation Method to the Stoichiometric Analysis of Fibrobacter succinogenes Growth by Erell Guiavarch; Agnes Pons; Catherine Creuly; Claude-Gilles Dussap (pp. 201-210).

Fibrobacter succinogenes S85, a strictly anaerobic Gram-negative bacterium, was grown in continuous culture in a bioreactor at different dilution rates (0.02 to 0.092 h⁻¹) on a fully synthetic culture medium with glucose as carbon source. Glucose and ammonium sulfate consumption, as well as biomass, succinate, acetate, formate, and carbohydrate production were regularly measured. The relevant biomass elemental compositions were established for each dilution rate. Robustness of the experimental information was checked by C and N mass balances estimation, which were satisfactory. A detailed overall stoichiometry analysis of the process, including all substrates and products of the culture, was proposed. Online and off-line parameters measured during the culture brought a large number of data which were weighted by their respective variance associated to the measured value. The material balance resulted in an overdetermined linear system of equations made of weighted relationships including experimental data, elemental balances (C, H, O, N, S, Na), and an additional constraint. The mass balances involved in stoichiometric equations were solved using data reconciliation and linear algebra methods to take into account error measurements. This methodology allowed to establish the overall stoichiometric equation for each dilution rate studied.

Keywords: Fibrobacter succinogenes ; Stoichiometry; Data reconciliation; Continuous culture; Mass balance

Regulation of Autolysis in Aspergillus nidulans by Tamás Emri; Zsolt Molnár; Melinda Szilágyi; István Pócsi (pp. 211-220).

In terms of cell physiology, autolysis is the centerpiece of carbon-starving fungal cultures. In the filamentous fungus model organism Aspergillus nidulans, the last step of carbon-starvation-triggered autolysis was the degradation of the cell wall of empty hyphae, and this process was independent of concomitantly progressing cell death at the level of regulation. Autolysis-related proteinase and chitinase activities were induced via FluG signaling, which initiates sporulation and inhibits vegetative growth in surface cultures of A. nidulans. Extracellular hydrolase production was also subjected to carbon repression, which was only partly dependent on CreA, the main carbon catabolite repressor in this fungus. These data support the view that one of the main functions of autolysis is supplying nutrients for sporulation, when no other sources of nutrients are available. The divergent regulation of cell death and cell wall degradation provides the fungus with the option to keep dead hyphae intact to help surviving cells to absorb biomaterials from dead neighboring cells before these are released into the extracellular space. The industrial significance of these observations is also discussed in this paper.

Keywords: Aspergillus nidulans ; Autolysis; Apoptosis; Chitinase; Proteinase; chiB; prtA; creA; fluG

Production and Purification of a Solvent-Resistant Esterase from Bacillus licheniformis S-86 by Sebastián Torres; Mario D. Baigorí; Ashok Pandey; Guillermo R. Castro (pp. 221-232).

New thermophilic and organic-solvent-tolerant Bacillus licheniformis S-86 strain is able to produce two active and solvent-stable esterases. Production of type I and II esterases was substantially enhanced when oils and surfactants were supplied as carbon sources. Grape oil (0.1% v/v) and Tween 20 to 60 (0.1% v/v) had enhanced enzyme production between 1.6- and 2.2-folds. Type II esterase was purified to homogeneity in a five-step procedure. This esterase was purified 76.7-fold with a specific activity of 135 U mg⁻¹. Molecular mass of the enzyme was estimated to be 38.4 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Type II esterase was active mostly on esters with short acyl chains, which allowed to classify the enzyme as a carboxylesterase with a K _m of 80.2 mmol l⁻¹ and a V _max of 256.4 μmol min⁻¹ mg⁻¹ for p-nitrophenyl acetate. Also, B. licheniformis S-86 type II esterase displayed activity in presence of water-miscible organic solvents at 50% concentration and stability after 1-h incubation.

Keywords: Esterases; B. licheniformis ; Production; Purification; Enzyme characterization; Detergents; Oils

Improving Specific Activity of Aspergillus carbonarius Polygalacturonase Using Polymeric Membranes by E. Nakkeeran; R. Subramanian; S. Umesh Kumar (pp. 233-243).

Microfiltration (MF) and ultrafiltration (UF) membranes were screened for improving the specific activity of polygalacturonases (PG) in the culture broth of Aspergillus carbonarius obtained after submerged fermentation. While 200 and 450 nm MF membranes eliminated some of the larger non-enzymatic proteins, 50 kDa UF membrane exhibited a marginal selectivity between the enzyme and other smaller proteins. The 450 nm MF and 50 kDa UF membranes selected were further evaluated under different process conditions for an integrated membrane process. The process efficacy of three different schemes was also studied for enzyme purification. A two-stage membrane process employing MF followed by UF improved the enzyme-specific activity (5,590 U/mg) by 4.69-fold eliminating the larger and smaller non-enzymatic proteins as well as non-protein impurities with a recovery of 76% enzymes, besides resulting in higher productivity. Thus, adoption of integrated membrane process with appropriate selection of membranes could result in high recovery of enzymes with improved specific activity.

Keywords: Aspergillus carbonarius ; Polygalacturonase; Downstream processing; Microfiltration; Ultrafiltration; Diafiltration; Integrated membrane process

Evaluation of the Probiotic Characteristics of Newly Isolated Lactic Acid Bacteria by Ravindran Girija Aswathy; Bindhumol Ismail; Rojan Pappy John; Kesavan Madhavan Nampoothiri (pp. 244-255).

Lactic acid bacteria were isolated from fermented vegetables, sour dough, milk products, sheep and human excreta. The newly isolated cultures were evaluated for a number of probiotic characteristics like bile salt resistance, salt tolerance in general, survival in low pH, hydrophobicity of the cell surface, resistance to low phenol concentration, antimicrobial activity and susceptibility pattern against vancomycin and erythromycin. The selected cultures were further screened for their ability to produce the nutraceticals such as folic acid and exopolysaccharide (EPS). Two potent isolates, CB2 (from cabbage) and SD2 (from sour dough) were found to produce both extracellular and intracellular folate. One of the isolates from yogurt (MC-1) and the one from whey (W3) produced significant amount of EPS with a maximum production of 8.79 ± 0.05 g/l by MC-1.

Keywords: Lactic acid bacteria; Probiotic; Nutraceutical; Exopolysaccharide; Folic acid

Detection of Virulence Genes in Vibrio cholerae Isolated from Aquatic Environment in Kerala, Southern India by Praveen Kumar; Wilson A. Peter; Sabu Thomas (pp. 256-262).

Vibrio cholerae is the etiologic agent of cholera. It is an autochthonous inhabitant of all aquatic environments. The virulence of V. cholerae is maintained by the CTX genetic element and tcpA gene. In the present investigation, environmental strains of V. cholerae isolated from different aquatic biotopes in Kerala were identified and serotyped. The antibiotic resistance pattern and presence of virulence and regulatory genes were examined. We found the presence of toxigenic non-O1/non-O139 strains harboring the CTX genetic element, heat-stable enterotoxin, rtxA gene, El Tor hemolysin, and Vibrio pathogenicity island (VPI). The strains also produced the cholera toxin (CT) as determined by monosialoganglioside enzyme-linked immunosorbent assay. A few strains belonging to the O1 serogroup but lacking the CTX genetic element were also observed. The majority of the environmental strains belonged to non-O1/non-O139 serogroup with many possessing toxR, ompU, heat-stable enterotoxin, and rtxA gene. The toxigenic non-O1/non-O139 strains exhibited resistance to trimethoprim, ampicillin, and polymixin B and intermediate resistance to co-trimoxazole. However, all other environmental strains were found resistant to ampicillin and polymixin B. Our findings demonstrate that the virulence genes are dispersed among the environmental strains of V. cholerae and a complex aquatic environment can give rise to pathogenic V. cholerae.

Keywords: Vibrio cholerae ; Cholera toxin; Toxin-coregulated pilus; RTX gene; Heat-stable enterotoxin

Extraction and Purification of Ipomoea Peroxidase Employing Three-phase Partitioning by A. V. Narayan; M. C. Madhusudhan; K. S. M. S. Raghavarao (pp. 263-272).

Three-phase partitioning (TPP) is a novel separation process used for the extraction and purification of biomolecules. The biomolecules are recovered in a purified form at the interface (precipitate), while the contaminants partition in t-butanol and aqueous phases. Peroxidase from the leaves of Ipomoea palmata was purified by using TPP. The ratio of the crude extract to t-butanol of 1:1 and 30% ammonium sulfate at 37 °C resulted in about 160% activity recovery and twofold purification in the aqueous phase of the first cycle of TPP. On subjecting the aqueous phase to the second cycle of TPP, a purification of 18-fold was achieved with about 81% activity recovery. The sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis showed substantial purification, and the molecular weight of peroxidase was found to be 20.1 KDa. The present study shows a higher degree of purification and activity yield as a primary purification process in comparison with existing literature values, thus demonstrating TPP as an attractive downstream process for the purification of peroxidase.

Keywords: Three-phase partitioning; Ipomoea palmata ; Peroxidase; Cross-partitioning; Aqueous two-phase extraction; Downstream process; t-Butanol; Ammonium sulfate

Construction of Protein Chip to Detect Binding of Mitf Protein (Microphthalmia Transcription Factor) and E-box DNA by Sang-Hee Yang; Jung-Sun Han; Seung-Hak Baek; Eun-Young Kwak; Hae Jong Kim; Jeong-Hyun Shin; Bong-Hyun Chung; Eun-Ki Kim (pp. 273-282).

A protein chip was constructed to detect the binding of microphthalmia-associated transcription factor (Mitf) and E-box DNA. Mitf, a key regulatory transcriptional factor of pigmentation-related genes such as tyrosinase, binds to specific sequence (CATGTG) in E-box DNA within the promoter of tyrosinase in the melanocytes. We produced Mitf as a maltose-binding protein (MBP) fusion protein in Escherichia coli, purified it using an affinity column, and immobilized it on β-cyclodextrin-coated glass plate. Binding of Mitf to its target DNA, E-box oligomer, was monitored by surface plasmon resonance (SPR), SPR imaging (SPRi), and fluorescence-based system. Among these detection methods, fluorescence method was the most reliable. In this method, fluorescent intensity was proportional to the DNA concentration (up to 20 μM) and Mitf (up to 500 μg/ml). Kinetics of DNA binding with Mitf showed Langmuir isotherm, and its kinetic constants were determined. It is expected that Mitf-E-box DNA chip can be used as a screening tool for depigmenting agents in the cosmetic industry.

Keywords: Mitf; E-box; Protein chip; HTS

Production and Characterization of the Exopolysaccharides Produced by Agaricus brasiliensis in Submerged Fermentation by L. F. O. Lima; S. Habu; J. C. Gern; B. M. Nascimento; Jose-Luis Parada; M. D. Noseda; A. G. Gonçalves; V. R. Nisha; Ashok Pandey; Vanete Thomaz Soccol; Carlos R. Soccol (pp. 283-294).

The aim of the work was to study the production of the exopolysaccharides by Agaricus brasiliensis and the isolation of exopolysaccharides (EPSs) with biological effects. A brasiliensis LPB03 was cultured in submerged fermentation in a medium containing glucose, yeast extract, hydrolyzed soybean protein, and salts (pH 6.1) at 29 °C and 120 rpm for 144 h. The maximum biomass and EPS yield was 7.80 ± 0.01 and 1,430.70 ± 26.75 mg/L, respectively. To isolate the produced EPSs, two methods were compared: (1) with alcohol precipitation and (2) treatment with tricloroacetic acid (TCA), followed by alcohol precipitation. The use of TCA facilitated the purification of the EPS, reducing the amount of the contaminant soy proteins. For monosaccharide identification, the EPSs were hydrolyzed, derivatized to alditol acetates, and analyzed by gas chromatography (GC) and GC-mass spectrometry, which showed the presence (in molar percentage) of mannose (58.7), galactose (21.4), and glucose (13.1) as major sugars, with lower amounts of rhamnose (3.9) and xylose (2.8). Scanning electron microscopy was used to observe the morphological structure of the EPS. The experiments in vivo including EPS in the mice diet during 8 weeks indicated the hipocholesteremic and hypoglycemic effects.

Keywords: Agaricus brasiliensis ; Submerged fermentation; Exopolysaccharide; Monossacharides composition; Biological properties

BTX Removal from Polluted Water Through Bioleaching Processes by Mehrdad Farhadian; David Duchez; Cédric Vachelard; Christian Larroche (pp. 295-306).

In this study, benzene, toluene, and xylenes (BTX) removal from contaminated water by physical, chemical, and biological processes was studied. Results showed that air sparging in polluted water can reduce monoaromatic compounds from 140,000 to about 5 μg/l in only 1 h process with a gassing rate of 0.33 VVM. This method cannot be considered as a green technology as pollutants are only transferred from the liquid phase to the gas phase The ultimate objective of this research was thus to evaluate the efficiency of a strategy involving BTX adsorption by granular-activated charcoal (GAC) and subsequent regeneration of this support by a bioleaching process. Analysis of such processes requires the building of analytical tools able to accurately determine the contents of the contaminants in samples containing biomass to make possible the calculation of reliable material balances. Current investigation showed that BTX are readily trapped by GAC particles with low further release in the liquid medium whereas they remain at least partially available for in situ biodegradation. BTX adsorption onto the GAC was shown to reach maximum solute retention close to 350, 250, and 150 (as mg/g GAC) for xylenes, toluene, and benzene, respectively. This approach, which could afford efficient biological active carbon regeneration, is very promising for the removal of BTX compounds from water without any further environment damage.

Keywords: Monoaromatic; Bioleaching; Contaminated water; Air sparging; GAC; BTX; Adsorption

Production of Biopesticides in an In Situ Cell Retention Bioreactor by Gunjan Prakash; Ashok K. Srivastava (pp. 307-318).

The seeds of Azadirachta indica contain azadirachtin and other limonoids, which can be used as a biopesticide for crop protection. Significant variability and availability of seed only in arid zones has triggered biotechnological production of biopesticides to cope up with its huge requirement. Batch cultivation of A. indica suspension culture was carried out in statistically optimized media (25.0 g/l glucose, 5.7 g/l nitrate, 0.094 g/l phosphate and 5 g/l inoculum) in 3 l stirred tank bioreactor. This resulted in 15.5 g/l biomass and 0.05 g/l azadirachtin production in 10 days leading to productivity of 5 mg l⁻¹ day⁻¹. Possible inhibition by the limiting substrates (C, N, P) were also studied and maximum inhibitory concentrations identified. The batch kinetic/inhibitory data were then used to develop and identify an unstructured mathematical model. The batch model was extrapolated to simulate continuous cultivation with and without cell retention in the bioreactor. Several offline computer simulations were done to identify right nutrient feeding strategies (with respect to key limiting substrates; carbon, nitrate and phosphate) to maintain non-limiting and non-inhibitory substrate concentrations in bioreactor. One such continuous culture (with cell retention) simulation was experimentally implemented. In this cultivation, the cells were propagated batch-wise for 8 days. It was then converted to continuous cultivation by feeding MS salts with glucose (75 g/l), nitrate (10 g/l), and phosphate (0.5 g/l) at a feed rate of 500 ml/day and withdrawing the spent medium at the same rate. The above continuous cultivation (with cell retention) demonstrated an improvement in cell growth to 95.8 g/l and intracellular accumulation of 0.38 g/l azadirachtin in 40 days leading to an overall productivity of 9.5 mg l⁻¹ day⁻¹.

Keywords: Bio-pesticide; Azadirachtin; Continuous cultivation; Mathematical modeling; Cell retention

Improving Performance of MFC by Design Alteration and Adding Cathodic Electrolytes by G. S. Jadhav; M. M. Ghangrekar (pp. 319-332).

Performance of two microbial fuel cells (MFCs) was investigated under batch and continuous mode of operation using different cathodic electrolyte. The wastewater was supplied from the bottom port provided to the anode chamber in both the MFCs and the effluent left the anode chamber from the top port in MFC-1, whereas in MFC-2, the effluent exit was provided close to membrane. Stainless steel (SS) mesh anode was used in both the MFCs with surface area of 167 and 100 cm² in MFC-1 and MFC-2, respectively. Under batch mode and continuous mode of operation, these MFCs gave chemical oxygen demand removal efficiency more than 85% and about 68%, respectively. Under batch mode of operation, maximum power density of 39.95 and 56.87 mW/m² and maximum current density of 180.83 and 295 mA/m² were obtained in MFC-1 and MFC-2, respectively. Under continuous mode of operation, a reduction in power and current density was observed. Even with less surface area of the anode, MFC-2 produced more current (1.77 mA) than MFC-1 (1.40 mA). Among the cathodic electrolyte tested, these can be listed in decreasing order of power density as aerated KMnO₄ solution > KMnO₄ solution without aeration > aerated tap water > aerated tap water with NaCl.

Keywords: Cathodic electrolytes; Design alteration; MFC; Power density

Batch Fermentation Model of Propionic Acid Production by Propionibacterium acidipropionici in Different Carbon Sources by Jefferson Coral; Susan Grace Karp; Luciana Porto de Souza Vandenberghe; José Luis Parada; Ashok Pandey; Carlos Ricardo Soccol (pp. 333-341).

Propionic acid (PA) is widely used as additive in animal feed and also in the manufacturing of cellulose-based plastics, herbicides, and perfumes. Salts of propionic acid are used as preservative in food. PA is mainly produced by chemical synthesis. Nowadays, PA production by fermentation of low-cost industrial wastes or renewable sources has been an interesting alternative. In the present investigation, PA production by Propionibacterium acidipropionici ATCC 4965 was studied using a basal medium with sugarcane molasses (BMSM), glycerol or lactate (BML) in small batch fermentation at 30 and 36 °C. Bacterial growth was carried out under low dissolved oxygen concentration and without pH control. Results indicated that P. acidipropionici produced more biomass in BMSM than in other media at 30 °C (7.55 g l⁻¹) as well as at 36 °C (3.71 g l⁻¹). PA and biomass production were higher at 30 °C than at 36 °C in all cases studied. The best productivity was obtained by using BML (0.113 g l⁻¹ h⁻¹), although the yielding of this metabolite was higher when using glycerol as carbon source (0.724 g g⁻¹) because there was no detection of acetic acid. By the way, when using the other two carbon sources, acetic acid emerged as an undesirable by-product for further PA purification.

Keywords: Propionibacterium acidipropionici ; Propionic acid; Batch fermentation; Carbon sources; Sugarcane molasses; Glycerol; Lactate

Growth Parameter and Viability Modifications of Escherichia coli by Phenolic Compounds and Argentine Wine Extracts by María J. Rodríguez Vaquero; María C. Manca de Nadra (pp. 342-352).

The anti-bacterial effect of pure non-flavonoids gallic, vanillic, protocatechuic, and caffeic acids and flavonoids quercetin, rutin, and catechin and the effect of total polyphenols of three Argentinean wine varieties, Cabernet Sauvignon, Malbec, and Merlot, against Escherichia coli, microorganism frequently detected in fresh and processed foods, was investigated. The hydroxycinnamic derivate caffeic acid and the flavonoid quercetin were the more effective against E. coli. The polyphenol effect was ethanol independent. The E. coli decimal reduction times were 2.9, 2.1, and 0.65 h for Malbec wine and 2.8, 2.3, and 0.64 h for Merlot wine with respect to 1×, 2×, and 4× concentrated wine samples, respectively. For Cabernet Sauvignon wine, the values were 6.3, 3.7, and 1.28 h for 1×, 2×, and 4× concentrated samples, respectively. With clarified wines, the decimal reduction times were higher with values ranging from 15 to 18.4 h in the wine samples. So the phenolic compounds present in red wines could be considered as an interesting alternative to be used as natural preservative against pathogenic microorganisms.

Keywords: Phenolic compounds; Anti-bacterial activity; Wine; Growth inhibition; Escherichia coli

Optimization of Enzymatic Clarification of Sapodilla Juice: A Statistical Perspective by Nicemol Jacob; R. K. Sukumaran; P. Prema (pp. 353-363).

Response surface methodology (RSM) was employed to establish optimum conditions for enzymatic clarification of sapodilla juice. Polygalacturonase obtained from Streptomyces lydicus had been purified to homogeneity and was used for the treatment. The independent variables were temperature (30–45 °C), enzyme concentration (0.5–1.5 U), and treatment time (30–90 min), whose effects on viscosity and clarity of the juice were evaluated using a Box–Behnken design. Significant regression models describing the changes of viscosity and clarity with respect to the independent variables were obtained, with the coefficient of determination, R ² , greater than 0.8. Based on response surfaces and contour plots, the optimum conditions for clarifying sapodilla juice were enzyme concentration 1.15 U, incubation temperature 34 °C, and incubation time 70 min.

Keywords: Streptomyces lydicus ; Polygalacturonase; Sapodilla juice; Enzymatic clarification; Response Surface Methodology

New Method Showing the Influence of Matrix Components in Leuconostoc mesenteroides Biofilm Formation by S. Badel; C. Laroche; C. Gardarin; T. Bernardi; P. Michaud (pp. 364-370).

Studying biofilm formation and influence of the matrix composition was heavy because only old and long methods were employed up to now: confocal microscopy, fluorescent chemical markers, and/or dying techniques. In this context, an innovative tool, the BioFilm Ring Test®, was here employed to explore the role of exopolysaccharides, proteins, and nucleic acids in the formation of biofilm by Leuconostoc mesenteroides. The principle is to add magnetic particles in the culture medium. When a biofilm is formed, particles are unable to migrate in the media to form a ring when a magnet is brought nearer to the well. Therefore, culture media supplemented with proteases, glycanases, and/or nucleases allowed us to identify the involvement of these substances in L. mesenteroides biofilm formation. The results permitted to demonstrate that dextran, proteins, and nucleic acids are implied in biofilm formation.

Keywords: Biofilm; Magnetization; Matrix components; Exopolysaccharides; Leuconostoc mesenteroides

Enhanced Production of Laccase from Coriolus versicolor NCIM 996 by Nutrient Optimization Using Response Surface Methodology by Santhiagu Arockiasamy; Indira Packialakshmi Gurusamy Krishnan; Nimalanandan Anandakrishnan; Sabitha Seenivasan; Agalya Sambath; Janani Priya Venkatasubramani (pp. 371-379).

Plackett and Burman design criterion and central composite design were applied successfully for enhanced production of laccase by Coriolus versicolor NCIM 996 for the first time. Plackett and Burman design criterion was applied to screen the significance of ten nutrients on laccase production by C. versicolor NCIM 996. Out of the ten nutrients tested, starch, yeast extract, MnSO₄, MgSO₄·7H₂O, and phenol were found to have significant effect on laccase production. A central composite design was applied to determine the optimum concentrations of the significant variables obtained from Plackett–Burman design. The optimized medium composition for production of laccase was (g/l): starch, 30.0; yeast extract, 4.53; MnSO₄, 0.002; MgSO₄·7H₂O, 0.755; and phenol, 0.026, and the optimum laccase production was 6,590.26 (U/l), which was 7.6 times greater than the control.

Keywords: Laccase; Plackett and Burman design; Central composite design; Enzyme activity; Coriolus versicolor

Selection and Optimization of Bacillus atrophaeus Inoculum Medium and its Effect on Spore Yield and Thermal Resistance by Sandra Regina B. R. Sella; Regina Elizabete F. Dlugokenski; Belquis P. Guizelini; Luciana P. S. Vandenberghe; Adriane B. P. Medeiros; Ashok Pandey; Carlos Ricardo Soccol (pp. 380-392).

Bacillus atrophaeus’s spores are used as biological indicators to monitor sterilization processes and as a Bacillus anthracis surrogate in the development and validation of biosafety methods. The regular use of biological indicators to evaluate the efficiency of sterilization processes is a legal requirement for health services. However, its high cost hinders its widespread use. Aiming at developing a cost-effective inoculum medium, soybean molasses and nutrient-supplemented vinasse were evaluated for their effectiveness in solid-state fermentation (SSF). In biomass production, the results demonstrated that all tested compositions favor growth by providing the nutritional demands of the microorganism. Optimum casein peptone and soybean molasses concentration (1.0%, 2.5%, or 4.0%) was determined by a 2^(2–0) factorial experimental design. The results have showed a positive influence of peptone on biomass production. In order to define peptone final concentration (4.0% or 6.0%), a 2² factorial experimental design was used. An optimized medium containing 4.0% soybean molasses and 4.0% casein peptone was similar in performance to a synthetic control medium (tryptone soy broth) in dry-heat thermal-resistant spore production by SSF. An experiment performed under optimum SSF conditions resulted in 1.9 × 10¹⁰ CFU g⁻¹ dry matter with D _160 °C = 5.2 ± 0.2 min.

Keywords: Bacillus atrophaeus ; Inoculum medium; Soybean molasses; Soybean vinasse; Optimization; Biological indicator; Spores

Organic Phase Synthesis of Ethyl Oleate Using Lipases Produced by Solid-state Fermentation by Antonio Martínez-Ruiz; Hugo Sergio García; Gerardo Saucedo-Castañeda; Ernesto Favela-Torres (pp. 393-401).

This paper reports a study of the enzymatic esterification of oleic acid and ethanol. The reaction was catalyzed by lipases produced by solid-state fermentation with Rhizopus sp. Olive oil and perlite were used as an inducer and inert support, respectively. Synthesis of ethyl oleate was carried out in a 10-mL batch reactor with magnetic stirring. The effects of substrate ratios, biocatalyst concentration, and temperature on the reaction rate and conversion efficiency were evaluated. The highest reaction rate (1.64 mmol/L min) was reached with an oleic acid/ethanol mol ratio of 1:5 (oleic acid 50 mM:ethanol 250 mM) and 1 g of biocatalyst. Conversions approaching 100% were obtained after 60 min of reaction at 45 °C with n-hexane as a solvent. The initial reaction rate increased proportionally with respect to biocatalyst concentration, which suggests that the reaction rate was not controlled by mass transfer. The biocatalyst retained more than 80% of its catalytic activity after 7 months of storage at 4 °C. The results demonstrate that the biocatalyst produced by Rhizopus sp. in solid-state fermentation can be successfully used for ethyl oleate synthesis over short reaction periods under conditions when ethanol is in excess.

Keywords: Lipases; Solid-state fermentation; Biosynthesis; Organic solvents

Antimicrobial and Antineoplasic Activity of Pleurotus ostreatus by E. R. S. Wolff; E. Wisbeck; M. L. L. Silveira; R. M. M. Gern; M. S. L. Pinho; S. A. Furlan (pp. 402-412).

The objectives of this work were to evaluate the antimicrobial and antineoplasic activity of Pleurotus ostreatus DSM 1833. To study the antimicrobial activity, the following extracts were prepared: water infusion of the fresh fruiting bodies (B1), dehydrated fruiting bodies (B2), fresh mycelium (M1), and dehydrated mycelium (M2). Polysaccharides from the fresh mycelium were also obtained by water infusion followed by ethanol treatment (EP). The extracts were tested against Candida albicans, Escherichia coli, and Bacillus subtilis. To investigate the antineoplasic effect of P. ostreatus, the culture broth in natura, the extract from the culture broth (ECB), and the extract from the fruiting bodies were tested in female Swiss albino mice inoculated with the Ehrlich ascitic tumor (EAT). B1, B2, and M1 showed more than 50.0% inhibition against C. albicans. M2 presented a high degree of inhibition against E. coli (87.5%) and B. subtilis (57.5%), while EP was not effective. All the tested substances inhibited the development of EAT at levels near 70% when injected intraperitoneally in mice. The highest tumor inhibition (76%) was achieved for the treatment with ECB. The intragastric treatment did not promote any reduction in tumor cell development, independent of the test substance.

Keywords: Pleurotus ostreatus ; Antimicrobial activity; Antineoplasic activity; Solid cultivation; Submerged cultivation; Extraction processes

Fed-batch Production of Gluconic Acid by Terpene-treated Aspergillus niger Spores by Sumitra Ramachandran; Pierre Fontanille; Ashok Pandey; Christian Larroche (pp. 413-423).

Aspergillus niger spores were used as catalyst in the bioconversion of glucose to gluconic acid. Spores produced by solid-state fermentation were treated with 15 different terpenes including monoterpenes and monoterpenoids to permeabilize and inhibit spore germination. It was found that spore membrane permeability is significantly increased by treatment with terpenoids when compared to monoterpenes. Best results were obtained with citral and isonovalal. Studies were carried out to optimize spores concentration (10⁷–10¹⁰ spores/mL), terpene concentrations in the bioconversion medium and time of exposure (1–18 h) needed for permeabilization of spores. Fed-batch production of gluconate was done in a bioreactor with the best conditions [10⁹ spores/mL of freeze-thawed spores treated with citral (3% v/v) for 5 h] followed by sequential additions of glucose powder and pH-regulated with a solution containing 2 mol/L of either NaOH or KOH. Bioconversion performance of the spore enzyme was compared with the commercial glucose oxidase at 50, 60, and 70 °C. Results showed that the spore enzyme was comparatively stable at 60 °C. It was also found that the spores could be reutilized for more than 14 cycles with almost similar reaction rate. Similar biocatalytic activity was rendered by spores even after its storage of 1 year at −20 °C. This study provided an experimental evidence of the significant catalytic role played by A. niger spore in bioconversion of glucose to gluconic acid with high yield and stability, giving protection to glucose oxidase.

Keywords: Aspergillus niger ; Fungal spore; Gluconic acid; Glucose oxidase; Citral; Monoterpenes

Effect of Carbon and Nitrogen on the Cannibalistic Behavior of Bacillus subtilis by Subir Kumar Nandy; K. V. Venkatesh (pp. 424-432).

Bacillus subtilis is known to exhibit cannibalism under nutrient limitation to delay sporulation. Cells of B. subtilis in phosphate buffer solution (PBS) demonstrate an oscillatory behavior in cell number due to cannibalism. Since PBS did not contain any nutrients, the effect of carbon and nitrogen sources on the cannibalistic behavior is unclear. In this study, the effect of external carbon and nitrogen on the cannibalistic behavior of B. subtilis is presented. The studies demonstrated that when glucose as a carbon source was introduced into PBS in the absence of any other nutrients, the cannibalistic tendency was delayed. This delay increased with the increase in the amount of glucose present in the PBS. Thus, the cannibalism was observed to be very sensitive to the amount of carbon present in the medium. However, when the medium contained only ammonium sulfate as a nitrogen source and was devoid of any carbon, the effect on cannibalism was minimal. The study, therefore, demonstrated that cannibalism was more sensitive to carbon than nitrogen indicating that the phenomenon of cannibalism may be more dependent on the status of energy in the medium than on nitrogen assimilation.

Keywords: Bacillus subtilis ; Cannibalism; Glucose; Ammonium sulfate; MBRT; Endospore

Denitrification of Highly Alkaline Nitrate Waste Using Adapted Sludge by Pradip B. Dhamole; Rashmi R. Nair; Stanislaus F. D’Souza; S. S. Lele (pp. 433-440).

Uranium extraction and regeneration of ion exchange resin generates concentrated nitrate effluents (typically 500 to 10,000 ppm NO₃–N) that are highly alkaline in nature (pH 9.0 to 11.0). It is difficult to remove nitrate from such solutions using standard physiochemical and biological methods. This paper reports denitrification of such wastes using preadapted sludge (biomass), which was acclimatized to different influent pH (7.5 to 11.5) in a sequencing batch reactor (4 l) for 2 months. Performance of the developed consortia was studied under different pH (7.5 to 12). Biomass denitrified the synthetic wastewater containing 1,694 ppm NO₃–N at a pH of 10.5. Decrease in nitrite build up was observed at higher pH, which differs from the reported results. Kinetic analysis of the data showed that specific rate of nitrate reduction was highest (78 mg NO₃–N/g MLSS/h) at higher pH (10.5). This was attributed to the acclimatization process. Thus, high-strength nitrate wastewater, which was highly alkaline, was successfully treated using preadapted sludge.

Keywords: Denitrification; pH; Acclimatization; Kinetics

Rhizobacteria in Mycorrhizosphere Improved Plant Health and Yield of Banana by Offering Proper Nourishment and Protection against Diseases by Niteen V. Phirke; Raman M. Kothari; Sudhir B. Chincholkar (pp. 441-451).

The corporate R&D banana orchards of Musa paradisiaca (dwarf Cavendish AAA, var. shrimanti) on a medium black alluvial soil with low nutrients harboured diversified species of vesicular-arbuscular mycorrhizal (VAM) fungi. These fungi infected the roots severely (69.2%), showed elevated (69.8 g⁻¹ soil) spore density, increased soil bacterial density (245 × 10⁸ cfu g⁻¹), produced siderophores (58.2%) and reduced nematode population (2.3 g⁻¹) in the mycorrhizosphere of plants for integrated plant nutrition management (IPNM) system as compared to traditional treatment of applying chemical fertilisers alone and other test treatments. The interactions of plant roots with native VAM and local and applied rhizobacteria in the matrix of soil conditioner enabled proper nourishment and protection of crop in IPNM treatment as compared to traditional way. Hence, exploitation of plant growth promoting rhizobacteria through judiciously designed IPNM system revealed the (a) relatively increased banana productivity (21.6%, 76 MT ha⁻¹), (b) least occurrence of fusarial wilt and negligible evidence of Sigatoka, (c) saving of 50% chemical fertilisers and (d) permitted control over soil fertility in producer’s favour over traditional cultivation practices. These findings are discussed in detail.

Keywords: Relative banana productivity; Rhizobacteria; VAM; Soil conditioner; IPNM; Sustainable production practices (SPP); mycorrhizosphere

Biomolecules and Nutritional Quality of Soymilk Fermented with Probiotic Yeast and Bacteria by C. R. Rekha; G. Vijayalakshmi (pp. 452-463).

Soymilk was fermented with five isolates of probiotic lactic acid bacteria and in combination with probiotic yeast Saccharomyces boulardii. Nutritional profile like fat, protein, ash, pH, acidity, polyphenol, and protein hydrolysis were analyzed. Polyphenol content decreased from 265.88 to 119 μg/ml with different cultures. Protein hydrolysis ranged from 2.46 to 2.83 mmol l⁻¹ with different cultures. The antioxidant activity was assessed using different methods like 1, 1-diphenyl-2-picrylhydrazyl free radical-scavenging assay, inhibition of ascorbate autoxidation, and measurement of reducing activity. The activities varied with the starters used but, nevertheless, were significantly higher than those found in unfermented soymilk. Bioconversion of the isoflavone glucosides (daidzin + genistin) into their corresponding bioactive aglycones (daidzein + genistein) was observed during soymilk fermentation. Total glucosides in soyamilk were 26.35 mg/100 ml. In contrast, aglycones genistein and daidzein were quantitatively lesser accounting 2.91 mg/100 ml (genistein 1.17 mg/100 ml and daidzein 1.19 mg/100 ml). Soymilk fermented with probiotic cultures resulted in the reduction of glycosides ranging from 0.40 mg to 1.36 mg/100 ml and increase in aglycones ranging from 6.32 mg to 13.66 mg/100 ml.

Keywords: Antioxidant activity; Biomolecule; Isoflavones; Polyphenol; Probiotic; S. boulardii

Comparing the Efficacy of Chlorine, Chlorine Dioxide, and Ozone in the Inactivation of Cryptosporidium parvum in Water from Parana State, Southern Brazil by Juliana Tracz Pereira; Adriana Oliveira Costa; Márcia Benedita de Oliveira Silva; Wagner Schuchard; Silvia Cristina Osaki; Edilene Alcântara de Castro; Rosangela Clara Paulino; Vanete Thomaz Soccol (pp. 464-473).

In the present work, assays were performed to compare the efficacy of hypochlorous acid, chlorine dioxide, and ozone in the inactivation of Cryptosporidium oocyst in public water supply from Brazilian South conditions. Experiments were carried out in samples containing 2 × 10⁴ oocysts/ml of C. parvum purified from feces of experimentally contaminated calves. An in vitro excystation method was used to evaluate oocysts’ viability and to determine the inactivation rates of hypochlorous acid at 2 ppm, chlorine dioxide at 1, 2, and 5 ppm, and ozone at the doses of 0.18, 0.24, 0.36, 0.48, and 1.44 mg/l. By using hypochlorous acid, the maximum inactivation rate obtained was 49.04% after 120 min. Chlorine dioxide at 5 ppm inactivated 90.56% of oocysts after 90 min of contact. Ozone was the most effective product, rendering an inactivation of 100% with the concentration of 24 mg/l. Resistance of Cryptosporidium to the usual disinfectants and the need for more effective water treatments to prevent waterborne diseases in Brazil are discussed in this manuscript.

Keywords: Chlorine dioxide; Cryptosporidium parvum ; Disinfection methods; Hypochlorous acid; In vitro excystation; Ozone

Biological Treatment of Shrimp Aquaculture Wastewater Using a Sequencing Batch Reactor by C. Lyles; R. Boopathy; Q. Fontenot; M. Kilgen (pp. 474-479).

To improve the water quality in the shrimp aquaculture, a sequencing batch reactor (SBR) has been tested for the treatment of shrimp wastewater. A SBR is a variation of the activated sludge biological treatment process. This process uses multiple steps in the same tank to take the place of multiple tanks in a conventional treatment system. The SBR accomplishes equalization, aeration, and clarification in a timed sequence in a single reactor basin. This is achieved in a simple tank, through sequencing stages, which include fill, react, settle, decant, and idle. A laboratory scale SBR and a pilot scale SBR was successfully operated using shrimp aquaculture wastewater. The wastewater contained high concentration of carbon and nitrogen. By operating the reactor sequentially, viz, aerobic and anoxic modes, nitrification and denitrification were achieved as well as removal of carbon in a laboratory scale SBR. To be specific, the initial chemical oxygen demand (COD) concentration of 1,593 mg/l was reduced to 44 mg/l within 10 days of reactor operation. Ammonia in the sludge was nitrified within 3 days. The denitrification of nitrate was achieved by the anaerobic process and 99% removal of nitrate was observed. Based on the laboratory study, a pilot scale SBR was designed and operated to remove excess nitrogen in the shrimp wastewater. The results mimicked the laboratory scale SBR.

Keywords: Nitrification; Denitrification; Sequencing batch reactor; Shrimp wastewater; Organic carbon

Vermicomposting of Solid Waste Generated from Leather Industries Using Epigeic Earthworm Eisenia foetida by B. Ravindran; S. L. Dinesh; L. John Kennedy; G. Sekaran (pp. 480-488).

Animal fleshing (ANFL) generated as solid waste from tannery industries was vermicomposted using the epigeic earthworm Eisenia foetida. The mixing ratio of ANFL with cow dung and agricultural residues as feed mixtures was maintained to be 3:1:1 respectively during the vermicomposting experiments for 50 days. Vermicomposting resulted in the reduction of pH 6.74 and C:N ratio 15.5 compared to the control sample. A notable increase in earthworm biomass was also observed in the vermin bioreactor. The germination index of 84% for tomato seedlings (Lycopersicon esculentum cv. PKM1) was observed for the vermicomposted soil. Scanning electron microscope and Fourier transform infrared spectroscopy were recorded to identify the changes in surface morphology and functional groups in the control and vermicomposted samples. The results obtained from the present study indicated that the earthworm E. foetida was able to convert ANFL into nutrient-enriched products.

Keywords: Eisenia foetida ; Animal fleshing; Vermicomposting; C:N ratio; Lycopersicon esculentum cv. PKM1

Effect of α-Ketoglutarate on Monoclonal Antibody Production of Hybridoma Cell Lines in Serum-Free and Serum-Containing Medium by Suthasinee Nilsang; Ashok Kumar; Sudip Kumar Rakshit (pp. 489-501).

Process development and optimization for increase population growth and protein productivity in mammalian cell culture have been studied for many years. In this study, the behavior of hybridoma cells was investigated using six-well micro-titer plate systems with a working volume of 4 ml. Mouse hybridoma cell lines D2 and 2C83G2 were seeded in serum-free and serum-containing media and cultured for 8 days. α-Ketoglutarate is an integral component of the tricarboxylic acid (TCA) cycle and is produced from glutamine via glutamate. To study its effect on cell growth, metabolism, and monoclonal antibody (mAb) production, 2 mM α-ketoglutarate (pH 7.2) was added in both media at the beginning of the cultivation and in another set after 72 h. High cell density was observed in D2 cell culturing in serum-free medium, while 2C83G2 cell line showed high cell density in serum-containing medium. However, both cell lines cultured in serum-free medium gave viability above 70% when grown for 8 days. The supplement of 2 mM α-ketoglutarate supported cell growth and mAb production of both hybridoma cell lines in serum-free and serum-containing medium. The addition of α-ketoglutarate at the beginning of the batch cultivation gave better result in cell growth and mAb production as compared to α-ketoglutarate supplementation after 72 h. However, addition after 72 h was better than no addition at all. This indicates that α-ketoglutarate have a positive effect on production and release of antibody.

Keywords: Antibody production; α-Ketoglutarate; Serum-free medium; Hybridoma

Spermine-Induced Morphogenesis and Effect of Partial Immersion System on the Shoot Cultures of Banana by L. Venkatachalam; N. Bhagyalakshmi (pp. 502-511).

Contribution of exogenous polyamines (PAs) and polyamine-inhibitors on plantlet regeneration patterns of banana (cv. Nanjanagudu Rasabale-AAB) was studied and the performance of regenerated shoots in temporary immersion system was evaluated. The rhizome explants (without shoot bud) of in vitro shoots produced a mixture of embryogenic and nonembryogenic calli on modified MS medium. The analyses of endogenous pools of polyamines showed higher levels of PAs in embryogenic than in nonembryogenic calli. Supplementation of various levels of (10–50 μM) spermine (Spm), spermidine (Spd), and putrescine (Put) to cultures with secondary embryogenesis showed that about 50% of embryogenic calli rapidly produced secondary embryos only in the presence 40 μM Spm but not in other treatments. The crucial role of Spm was further confirmed by the use of 0.1 mM each of α-DL-Difluromethylornithine and α-DL-Difluromethylarginine along with Spm where the presence of inhibitors concomitantly inhibited the secondary embryogenesis. The shoots obtained from the embryogenic cultures were checked for their performance on solid medium (SM) and partial immersion system (PIS). The rate of shoot multiplication was higher in PIS than in SM throughout 6 weeks culture period. Uniformity in elongation of all the shoot buds was observed in PIS but not in SM. Evaluation for the acclimatization, survival under greenhouse conditions revealed the better performance of PIS-derived plants than those from SM.

Keywords: Embryogenesis; Musa ; Partial immersion system; Polyamine

Purification and Biochemical Characterization of Methionine Aminopeptidase (MetAP) from Mycobacterium smegmatis mc²155 by Sai Shyam Narayanan; Ajeena Ramanujan; Shyam Krishna; Kesavan Madhavan Nampoothiri (pp. 512-521).

The methionine aminopeptidase (MetAP) catalyzes the removal of amino terminal methionine from newly synthesized polypeptide. MetAP from Mycobacterium smegmatis mc² 155 was purified from the culture lysate in four sequential steps to obtain a final purification fold of 22. The purified enzyme exhibited a molecular weight of ≈ 37 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Activity staining was performed to detect the methionine aminopeptidase activity on native polyacrylamide gel. The enzyme was characterized biochemically, using l-methionine p-nitroanilide as substrate. The enzyme was found to have a temperature and pH optimum of 50 °C and 8.5, respectively, and was found to be stable at 50 °C with half-life more than 8 h. The enzyme activity was enhanced by Mg²⁺ and Co²⁺ and was inhibited by Fe²⁺ and Cu²⁺. The enzyme activity inhibited by EDTA is restored in presence of Mg²⁺ suggesting the possible role of Mg²⁺ as metal cofactor of the enzyme in vitro.

Keywords: Mycobacterium smegmatis mc² 155; Methionine aminopeptidases; l-methionine p-nitroanilide; Peptide processing; Metalloenzyme

Reverse Micellar Extraction of β-Galactosidase from Barley (Hordeum vulgare) by A. B. Hemavathi; H. Umesh Hebbar; K. S. M. S. Raghavarao (pp. 522-531).

The reverse micellar system of sodium bis(2-ethylhexyl)sulfosuccinate (AOT)/isooctane was used for the extraction and primary purification of β-galactosidase (EC 3.2.1.23) from the aqueous extract of barley (Hordeum vulgare) for the first time. The process parameters such as the concentration of the surfactant, the volume of the sample injected, and its protein concentration, pH, and ionic strength of the initial aqueous phase for forward extraction, buffer pH, and salt concentration for back extraction are varied to optimize the extraction efficiency. Studies carried out with both phase transfer and injection mode of reverse micellar extraction confirmed the injection mode to be more suitable for β-galactosidase extraction. The extent of reverse micellar solubilization of proteins increased with an increase in protein concentration of the feed sample. However, back extraction efficiency remained almost constant (13–14.4%), which indicates the selectivity of AOT reverse micelles for a particular protein under given experimental conditions. β-Galactosidase was extracted with an activity recovery of 98.74% and a degree of purification of 7.2-fold.

Keywords: Reverse micellar system; Barley; β-Galactosidase; Phase transfer; Injection mode

Biological Treatment of Toxic Petroleum Spent Caustic in Fluidized Bed Bioreactor Using Immobilized Cells of Thiobacillus RAI01 by Ravichandra Potumarthi; Gopal Mugeraya; Annapurna Jetty (pp. 532-546).

In the present studies, newly isolated Thiobacillus sp was used for the treatment of synthetic spent sulfide caustic in a laboratory-scale fluidized bed bioreactor. The sulfide oxidation was tested using Ca-alginate immobilized Thiobacillus sp. Initially, response surface methodology was applied for the optimization of four parameters to check the sulfide oxidation efficiency in batch mode. Further, reactor was operated in continuous mode for 51 days at different sulfide loading rates and retention times to test the sulfide oxidation and sulfate and thiosulfate formation. Sulfide conversions in the range of 90–98% were obtained at almost all sulfide loading rates and hydraulic retention times. However, increased loading rates resulted in lower sulfide oxidation capacity. All the experiments were conducted at constant pH of around 6 and temperature of 30 ± 5 °C.

Keywords: Autotrophic bacteria; Sulfide oxidation; Response surface methodology; Thiobacillus sp. ; Fluidized bed reactor; Optimization

Effect of Aeration on Production of Anticancer Lignans by Cell Suspension Cultures of Linum album by Ashish Baldi; Ashok K. Srivastava; Virendra S. Bisaria (pp. 547-555).

The effects of aeration within the range of 0.2–0.5 vvm on transformed and high yielding cell cultures of Linum album were investigated in a 5-L stirred tank bioreactor equipped with low shear Setric impeller. The kinetics of cell growth, substrate utilization, and production of lignans, namely, podophyllotoxin and 6-methoxypodophyllotoxin, were established. Maximum biomass of 23.2 g/L and lignan accumulation levels of 176.3 mg/L podophyllotoxin and 10.86 mg/L 6-methoxypodophyllotoxin were obtained with initial air flow rate of 0.3 vvm. Specified oxygen demand of cells was estimated to be 1.35 g O₂/g biomass. The optimum oxygen transfer coefficient was found to be 16.7 h⁻¹ _, which corresponded to aeration rate of 0.3 vvm. The effect of minimum dissolved oxygen (DO) concentration was investigated with respect to biomass and lignan production by comparing identically aerated and agitated bioreactor cultivations at dissolved oxygen concentrations of 10%, 30%, and 50%. Cell growth and podophyllotoxin accumulation were not affected significantly at these DO levels, but 6-methoxypodophyllotoxin production was enhanced when cells were cultivated at 30% DO level. The maximum volumetric productivities of 18.2 mg/L day and 3.2 mg/L day for podophyllotoxin and 6-methoxypodophyllotoxin, respectively, were obtained. These results establish the key role of oxygen on mass scale production of anticancer lignans by cell cultures of L. album. It may serve as a suitable parameter for scale-up.

Keywords: Aeration; Dissolved oxygen; Linum album ; 6-Methoxypodophyllotoxin; Podophyllotoxin; Stirred tank bioreactor

Dual Elicitation for Improved Production of Withaferin A by Cell Suspension Cultures of Withania somnifera by A. Baldi; D. Singh; Vinod K. Dixit (pp. 556-564).

High yielding transformed callus culture of W. somnifera was established by infecting hypocotyls with Agrobacterium tumefaciens MTCC-2250. Maximum withaferin A content of 0.0875 mg/g dry cell weight and transformation efficiency of 80% were obtained. Confirmation of transformation was done on the basis of the presence of the ags gene by using polymerase chain reaction. Various abiotic elicitors (arachidonic acid, methyl jasmonate, calcium chloride, and copper sulfate) and biotic elicitors (cell extracts and culture filtrates of Alternia alternata, Fusarium solani, and Verticilium dahaliae) were tested at different concentrations to enhance withaferin A production in suspension culture of transformed cells. Maximum enhancements of 5.4 times and 9.7 times, respectively, were obtained when copper sulfate (100 μM) and the cell extract of V. dahaliae (5% v/v) were added separately to suspension cultures. The dual elicitation strategy by the combined addition of these two elicitors resulted in 13.8-fold enhancement of withaferin A content in comparison to control cultures (2.65 mg/L). The present study indicates the potential of this biotechnology-based methodology for the large-scale production of withaferin A.

Keywords: Agrobacterium ; Biotic elicitor; Elicitation; Suspension culture; Withaferin A; Withania somnifera

Superoxide Dismutase-Loaded PLGA Nanoparticles Protect Cultured Human Neurons Under Oxidative Stress by Maram K. Reddy; Li Wu; Wei Kou; Anuja Ghorpade; Vinod Labhasetwar (pp. 565-577).

The objective of our study was to investigate the neuroprotective efficacy of superoxide dismutase (SOD), loaded in poly(d,l-lactide co-glycolide; PLGA) nanoparticles (NPs), in cultured human neurons challenged with hydrogen peroxide (H₂O₂)-induced oxidative stress. We hypothesized that the protected and sustained intracellular delivery of SOD encapsulated in NPs would demonstrate better neuroprotection from oxidative stress than either SOD or pegylated SOD (PEG-SOD) in solution. SOD-NPs (~81 ± 4 nm in diameter, 0.9% w/w SOD loading) released the encapsulated SOD in an active form with 8.2% cumulative release during the first 24 h, followed by a slower release thereafter. The results demonstrated that PLGA-NPs are compatible with human neurons, and the neuroprotective effect of SOD-NPs is dose-dependent, with efficacy seen at >100 U SOD, and less significant effects at lower doses. Neither SOD (25–200 U) nor PEG-SOD (100 U) in solution demonstrated the neuroprotective effect under similar conditions. The neuroprotective effect of SOD-NPs was seen up to 6 h after H₂O₂-induced oxidative stress, but the effect diminished thereafter. Confocal microscopic studies demonstrated better intracellular neuronal uptake of the encapsulated model protein (fluorescein isothiocyanate-labeled BSA) than the protein in solution. Thus, the mechanism of efficacy of SOD-NPs appears to be due to the stability of the encapsulated enzyme and its better neuronal uptake after encapsulation.

Keywords: Free radicals; Antioxidant enzymes; Drug delivery; Polymers; Sustained release

Effects of Organic Solvents on Immobilized Lipase in Pectin Microspheres by L. Costas; V. E. Bosio; A. Pandey; G. R. Castro (pp. 578-586).

Lipase from Brevibacillus agri 52 was found stable up to 90% diethylenglycol (DEG), glycerol (GLY), and 1,2 propanediol (1,2 PRO) at 37 °C for 1 h and the stability was reduced only approximately 20% after 12 h incubation, but in 40% dimethylsulfoxide (DMSO), lipase activity was stable only for 1 h. Inhibition of the biocatalysts with dimethylformamide (DMF) was detected at 20% solvent concentration. In water immiscible systems, the stability of lipase in n-hexane, n-tetradecane and n-heptane resembles the water activity, but in the presence of isobutanol, 1-hexanol, and butylbutirate, the stability was significantly reduced. Lipase 52 precipitates in the presence of 50% acetone or ethanol/water mixtures, but enzymatic activity was partially recovered by adding 20% GLY, DEG, 1,2 PRO, or DMSO to the reaction mixture. Furthermore, by increasing DEG in 70% DMF/DEG mixtures, the lipase activity was protected. Encapsulation of lipase in pectin gels cross-linked with calcium ions brings three to four times more enzymatic activity in 70% water miscible organic solvents compared to aqueous systems.

Keywords: Non-aqueous biocatalysis; Lipases; Enzyme stability; Solvent mixtures; Pectin gels; Gel microspheres; Enzyme encapsulation

Mediator-assisted Decolorization and Detoxification of Textile Dyes/Dye Mixture by Cyathus bulleri Laccase by Meenu Chhabra; Saroj Mishra; T. R. Sreekrishnan (pp. 587-598).

Laccase from basidiomycete fungus Cyathus bulleri was evaluated for its ability to decolorize a number of reactive and acidic dyes in the presence of natural and synthetic mediators. The extent of decolorization was monitored at different mediator/dye concentrations and incubation time. Among the synthetic mediators, 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) was effective at low mediator/dye ratios and resulted in 80–95% decolorization at rates that varied from 226 ± 4 nmol min⁻¹ mg⁻¹ for Reactive Orange 1 to 1,333 ± 15 nmol min⁻¹ mg⁻¹ for Reactive Red 198. Other synthetic mediators like 1-hydroxybenzotriazole and violuric acid showed both concentration- and time-dependent increases in percent decolorization. Natural mediators like vanillin, on the other hand, were found to be less effective on all the dyes except Reactive Orange 1. Computed rates of decolorization were about twofold lower than that with ABTS. The laccase–ABTS system also led to nearly 80% decolorization for the simulated dye mixture. No clear correlation between laccase activity on the mediator and its ability to decolorize dyes was found, but pH had a significant effect: Optimum pH for decolorization coincided with the optimum pH for mediator oxidation. The treated samples were also evaluated for toxicity in model microbial systems. The laccase–mediator system appears promising for treatment of textile wastewaters.

Keywords: Cyathus bulleri ; Dye decolorization; Mediator-assisted decolorization; Laccase; Detoxification

Fatty Acid Profiling During Microbial Lipid Production Under Varying pO₂ and Impeller Tip Speeds by Syed Ubaid Ahmed; Sudheer Kumar Singh; Ashok Pandey; Sanjit Kanjilal; Rachapudi B. N. Prasad (pp. 599-609).

The fatty acid profile study was undertaken to study the effect of impeller tip speed-associated shear stress and dissolved oxygen saturation (DO) on the fatty acid composition variation and on total lipid content of the cells. The study was undertaken in a 5-l stirred tank bioreactor using Mucor sp. RRL001. To study the interaction of parameters and their effects, a central composite design was used. The fatty acid profiling during the course of study suggested that oleic acid and palmitic acid were two major components with their composition varying between 34–47% and 29–39.1%, respectively, of the total lipid content. The GLA content varied between 3% and 9% of the total lipid. The lipid profile study also revealed the presence of a minor amount of fatty acids of chain length C:12, C:20, C:22, and C:24. The modeling of lipid accumulation suggested that it follows a quadratic model with both impeller tip speed (p = 0.0166) and dissolved oxygen concentration (p = 0.0098) following the quadratic order of effect. The fermenter run based on the optimum production zone in response surface plot resulted in the maximum 4.8 g l⁻¹ lipid compared with the model-predicted value of 4.49 g l⁻¹. The present study suggests that dissolved oxygen saturation is a more significant contributor to total lipid accumulation. However, the study also suggests that the fatty acid profile of fungal lipid is not directly associated with the shear stress or oxygen availability in Mucor sp. RRL001.

Keywords: Microbial lipid; Fatty acid profile; Central composite design; Impeller tip speed; Dissolved oxygen concentration

Lactase Production by Solid-state Cultivation of Kluyveromyces marxianus CDBBL 278 on an Inert Support: Effect of Inoculum, Buffer, and Nitrogen Source by Luz Tovar-Castro; Mariano García-Garibay; Gerardo Saucedo-Castañeda (pp. 610-617).

A study was carried out to select the conditions for cultivation of Kluyveromyces marxianus CDBBL 278 in solid-state culture (SSC) using polyurethane foam (PUF) as an inert support. PUF was impregnated with culture media containing lactose (50 g/L) as the carbon and energy source. Evaluation of culture parameters during different growth phases was carried out by respirometry. The effect of inoculum level, buffer capacity of the medium, and nitrogen source upon the yield of biomass on lactose (Yx/s) and production of lactase and inulinase was investigated. The highest lactase titre was achieved with an inoculum level of 1 × 10⁷ cells per gram of wet matter (gwm) and 20% of the total nitrogen source provided as urea. The best biomass yield (0.37) was obtained when less than 40% of the total nitrogen was provided as urea. Using potassium phosphate allowed 90% substrate consumption in 30 h. In the best conditions, intracellular lactase and extracellular inulinase activities of 1147.7 IU/gX and 241.6 IU/gX were obtained, respectively, with a lag phase of 13.8 h and a rate of respiratory activity (μ _CO2) of 0.23 ± 0.01 h⁻¹. To our knowledge, this is the first report on lactase production by K. marxianus CDBBL 278 in SSC. This study gives basic information about biomass yield and enzyme production using lactose as the sole carbon source in SSC on an inert support.

Keywords: Kluyveromyces marxianus ; Solid-state culture; Enzymatic activities

Remediation of Textile Dye Waste Water Using a White-Rot Fungus Bjerkandera adusta Through Solid-state Fermentation (SSF) by Tim Robinson; Poonam Singh Nigam (pp. 618-628).

A strict screening strategy for microorganism selection was followed employing a number of white-rot fungi for the bioremediation of textile effluent, which was generated from one Ireland-based American textile industry. Finally, one fungus Bjerkandera adusta has been investigated in depth for its ability to simultaneously degrade and enrich the nutritional quality of highly coloured textile effluent-adsorbed barley husks through solid-state fermentation (SSF). Certain important parameters such as media requirements, moisture content, protein/biomass production and enzyme activities were examined in detail. A previously optimised method of dye desorption was employed to measure the extent of dye remediation through effluent decolorisation achieved as a result of fungal activity in SSF. B. adusta was capable of decolourising a considerable concentration of the synthetic dye effluent (up to 53%) with a moisture content of 80–85%. Protein enrichment of the fermented mass was achieved to the extent of 229 g/kg dry weight initial substrate used. Lignin peroxidase and laccase were found to be the two main enzymes produced during SSF of the dye-adsorbed lignocellulosic waste residue.

Keywords: Barley husks; Textile dyes; Bjerkandera adusta ; Laccase; Lignin peroxidase; Manganese peroxidase; Decolourisation; Solid-state fermentation (SSF)

Enriching Oryzanol in Rice Bran Oil using Membranes by S. Manjula; R. Subramanian (pp. 629-637).

Oryzanol present in rice bran is associated with various physiological functions. However, these beneficial ferulate esters are lost to the extent of 87% during conventional refining of crude rice bran oil. In the present investigation, oryzanol enrichment in rice bran oil was attempted using nonporous polymeric membranes under undiluted as well as hexane-diluted conditions with different (crude, refined, and model oil) systems varying widely in their oryzanol content. During membrane processing, oryzanol content in the refined rice bran oil increased from 2,420 to 7,340 mg/kg (approximately threefold enrichment). While processing crude oil and model oil systems, the oryzanol content in the oil improved from 17,600 to 27,300 mg/kg and 20,400 to 30,300 mg/kg, respectively. The enrichment of oryzanol was due to its moderate rejection by the nonporous hydrophobic membrane owing to the hydrophilic nature of the ferulic esters. Hexane dilution improved the oil flux by one order of magnitude but reduced the selectivity. Enriched rice bran oil may find wider applications in the pharmaceutical, therapeutic, and dietary preparations as well as in producing standard cooking oil with guaranteed oryzanol content.

Keywords: Enrichment; Flux; Hexane; Nonporous membrane; Oleic acid; Oryzanol; Rejection; Rice bran oil

Functional Gene Abundances (nahAc, alkB, xylE) in the Assessment of the Efficacy of Bioremediation by Jani M. Salminen; Pirjo M. Tuomi; Kirsten S. Jørgensen (pp. 638-652).

In this study, we compared the mineralization rates of three selected ¹⁴C-labeled hydrocarbon compounds, octacosane, toluene, and naphthalene, with the presence of the corresponding functional genes (alkB, xylE, nahAc) in a large number of soil samples representing different types of soil and petroleum hydrocarbon contamination. Functional genes were enumerated by the replicate limited dilution (RLD) polymerase chain reaction (PCR) technique. RLD-PCR was further compared to real-time PCR measurements for nahAc and xylE for some samples. At a heating oil-contaminated site, octacosane mineralization rates were higher (on average 0.0015 day⁻¹) when compared to aerobic naphthalene and toluene mineralization (on average 0.00003 and 0.0007 day⁻¹). The corresponding gene abundances measured by RLD-PCR were on average 0.95, 0.3, and 0.13 × 10³ gene copies g⁻¹ soil for alkB, nahAc, and xylE, respectively. At a site contaminated with gasoline, the situation was the opposite: Toluene mineralization was the highest (on average 0.0031 day⁻¹), and only xylE genes could be detected (on average 0.13 × 10³ gene copies g⁻¹ soil by RLD-PCR). XylE and nahAc gene abundances were correlated with the ¹⁴C-toluene and naphthalene mineralization activities, respectively, in samples from aerobic layers. AlkB gene abundances were not correlated with the octacosane mineralization. Real-time PCR was a more sensitive method than RLD-PCR by a factor of 1,200 for nahAc and 300 for xylE. In conclusion, functional gene abundances seemed to reflect the type of the contamination. With optimized assays, the gene abundances can be used to assess bioremediation efficacy.

Keywords: Functional gene; alkB ; nahAc ; xylE ; Oil-contaminated sites; Bioremediation

Cephalosporins Determination with a Novel Microbial Biosensor Based on Permeabilized Pseudomonas aeruginosa Whole Cells by Sanjay Kumar; Subir Kundu; K. Pakshirajan; V. Venkata Dasu (pp. 653-664).

A new potentiometric microbial biosensor based on Pseudomonas aeruginosa was developed in this study for detecting the cephalosporin group of antibiotics. Preliminary results with the biosensor indicated that P. aeruginosa cells, when treated with lysozyme, showed more efficiency in detecting cephalosporin C in a wide concentration range of 0.1–11 mM with high sensitivity compared to the normal cells. Optimization of the three important biosensor design parameters permeabilized cell contents, quantities of gelatin, and glutaraldehyde resulted in high performance of the biosensor. The optimized values of the above parameters were cell contents 2.5 mg/cm², gelatin 8.5 mg/cm², and 0.25% glutaraldehyde. The assay conditions, namely phosphate buffer pH, ionic strength, and temperature, were optimized for best performance of the biosensor. The specificity test of the biosensor towards detecting different β-lactam antibiotics showed good response only for the cephalosporins. The operational and storage stability in detecting cephalosporin C indicated very good potential of the biosensor in detecting cephalosporins with high accuracy.

Keywords: Pseudomonas aeruginosa ; Microbial biosensor; Cephalosporins; β-Lactamase; Cephalosporinase; Permeabilized cells

Exploring Antagonistic Metabolites of Established Biocontrol Agent of Marine Origin by Makarand Ramesh Rane; Prashant Diwakar Sarode; Bhushan Liladhar Chaudhari; Sudhir Bhaskarrao Chincholkar (pp. 665-675).

Biocontrol ability of Pseudomonas aeruginosa ID 4365, a biocontrol agent of groundnut phytopathogens from marine origin, was previously attributed to the production of pyoverdin type of siderophores. However, pyoverdin-rich supernatants of this organism showed better antifungal activity compared to equivalent amount of purified pyoverdin indicating presence of undetected metabolite(s) in pyoverdin rich supernatants. On the basis of observation that antagonistic activity was iron-dependent and iron-independent, an attempt was made to detect the presence of additional metabolites. In addition to pyoverdin, strain produced additional siderophores, viz. pyochelin and salicylic acid. Two broad spectrum antifungal compounds, viz. pyocyanin and phenazine-1-carboxylic acid, were detected, characterized, and activity against phytopathogens was demonstrated. Iron- and phosphate-dependent co-production of siderophores and phenazines was confirmed. Strain showed additional features like production of hydrogen cyanide, indol-3-acetic acid, and phosphate solubilization.

Keywords: Biocontrol agent; Pseudomonas ; PGPR; Phenazines; Siderophores

Some Methods for Human Liquid and Solid Waste Utilization in Bioregenerative Life-Support Systems by S. A. Ushakova; I. G. Zolotukhin; A. A. Tikhomirov; N. A. Tikhomirova; Yu. A. Kudenko; I. V. Gribovskaya; Yu. Balnokin; J. B. Gros (pp. 676-685).

Bioregenerative life-support systems (BLSS) are studied for developing the technology for a future biological life-support system for long-term manned space missions. Ways to utilize human liquid and solid wastes to increase the closure degree of BLSS were investigated. First, urine and faeces underwent oxidation by Kudenko’s physicochemical method. The products were then used for root nutrition of wheat grown by the soil-like substrate culture method. Two means of eliminating sodium chloride, introduced into the irrigation solution together with the products of urine oxidation, were investigated. The first was based on routine electrodialysis of irrigation water at the end of wheat vegetation. Dialysis eliminated about 50% of Na from the solution. This desalinization was performed for nine vegetations. The second method was new: after wheat cultivation, the irrigation solution and the solution obtained by washing the substrate containing mineral elements not absorbed by the plants were used to grow salt-tolerant Salicornia europaea L. plants (saltwort). The above-ground biomass of this plant can be used as a food, and roots can be added to the soil-like substrate. Four consecutive wheat and Salicornia vegetations were cultivated. As a result of this wheat and Salicornia cultivation process, the soil-like substrate salinization by NaCl were considerably decreased.

Keywords: Life-support systems; Closure; Human waste; Sodium chloride; Salicornia

Dynamic Aspects and Controllability of the MELiSSA Project: A Bioregenerative System to Provide Life Support in Space by Bérangère Farges; Laurent Poughon; Catherine Creuly; Jean-François Cornet; Claude-Gilles Dussap; Christophe Lasseur (pp. 686-699).

Manmade ecosystems differ from their prototype biosphere by the principle of control. The Earth Biosphere is sustainable by stochastic control and very large time constants. By contrast, in a closed ecosystem such as the micro-ecological life support system alternative (MELiSSA system) developed by the European Space Agency for space exploration, a deterministic control is a prerequisite of sustainable existence. MELiSSA is an integrated sum of interconnected biological subsystems. On one hand, all unit operations in charge of the elementary functions constitutive of the entire life support system are studied until a thorough understanding and mathematical modelling. On the other hand, the systemic approach of complex, highly branched systems with feedback loops is performed. This leads to study in the same perspective, with the same degree of accuracy and with the same language, waste degradation, water recycling, atmosphere revitalisation and food production systems prior to the integration of knowledge-based control models. This paper presents the mathematical modelling of the MELiSSA system and the interface between the control strategy of the entire system and the control of the bioreactors.

Keywords: Bioregenerative life support; Closed system; Predictive control; Mathematical modelling

Effect of Aeration and Agitation Regimes on Lipase Production by Newly Isolated Rhodotorula mucilaginosa–MTCC 8737 in Stirred Tank Reactor Using Molasses as Sole Production Medium by Ravichandra Potumarthi; Chennupati Subhakar; J. Vanajakshi; Annapurna Jetty (pp. 700-710).

The influence of media and process parameters (aeration and agitation) on fermentation broth rheology and biomass formation has been studied in 1.5-l stirred tank reactor for lipase production using Rhodotorula mucilaginosa MTCC 8737. Molasses, as sole production medium, is used for lipase production by varying aeration (1, 2, and 3 vvm) and agitation speeds (100, 200, and 300 rpm). Maximum lipase activity of 72 U/ml was obtained during 96 h of fermentation at 2 vvm, 200 rpm, pH 7, and 25 ± 2°C temperature. Lipase production kinetics with respect to dry cell weight of biomass showed Y _P/S of 25.71 U/mg, specific product formation of 10.9 U/mg DC, and Y _X/S 2.35 mg/mg. Maximum lipase activity (MC 2) of 56 U/ml was observed at 1% molasses, and a further increase in the molasses concentration of (%) 1.5 and 2 inhibited the product formation of lipase with 15 and 8.5 U/ml, respectively. The production kinetics of molasses media showed Y _P/X was 14 U/mg DC, Y _P/S 16 U/mg, and Y _X/S 1.14 mg/mg during 96 h of bioreactor operation. The k_La values for all batches (MC 1–MC 4) at 96 h of fermentation were 32, 28, 21, and 19/h, and the |oxygen transfer rate were 54.4, 56, 35.7, and 17.29 mg/l h, respectively. Increase in molasses concentration resulted in decreased lipase activity by increase in viscosity of the fermentation broth.

Keywords: Aeration; Agitation; Oxygen transfer rate; Lipase activity; Viscosity

A New Microplate Screening Method for the Simultaneous Activity Quantification of Feruloyl Esterases, Tannases, and Chlorogenate Esterases by L. Ramírez; J. Arrizon; G. Sandoval; A. Cardador; R. Bello-Mendoza; P. Lappe; J. C. Mateos-Díaz (pp. 711-723).

Feruloyl, chlorogenate esterases, and tannases are enzymes useful in phenolic modifications of pharmaceutical relevance as protectors against several degenerative human diseases. Therefore, there is a growing interest in discovering new sources of these enzymes. However, traditional methods for their activity measurements are time-consuming and poorly adapted for high-throughput screening. In this study, a successful new microplate high-throughput screening method for the simultaneous quantification of all mentioned activities is demonstrated. This method allows the detection of activities as low as 1.7 mU ml⁻¹. Furthermore, reaction rates increased proportionally with the amount of enzyme added, and no interferences with the other commercial hydrolases tested were found. The utility of the method was demonstrated after simultaneously screening feruloyl, chlorogenate esterase, and tannase activities in solid state fermentation extracts obtained during the kinetics of production of 20 fungal strains. Among these, seven strains were positive for at least one of the esterase activities tested. This result shows the potential for the rapid routine screening assays for multiple samples of moderate low to high enzymatic levels.

Keywords: High-throughput screening; Esterases; Coffee wastes; Solid state fermentation

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.151, #2-3)

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.151, #2-3)