Aerobic biodegradation of nitrobenzene by a defined microbial consortium immobilized in polyurethane foam

An aerobic microbial consortium constructed by the combination of Rhodotorula mucilaginosa Z1, Streptomyces albidoflavus Z2 and Micrococcus luteus Z3 was immobilized in polyurethane foam and its ability to degrade nitrobenzene was investigated. Batch experimental results showed that polyurethane-foam-immobilized cells (PFIC) more efficiently degrade 200–400 mg l⁻¹ nitrobenzene than freely suspended cells (FSC). Kinetics of nitrobenzene degradation by PFIC was well described by the Andrews equation. Compared with FSC, PFIC exhibited better reusability (over 100 times) and tolerated higher shock-loadings of nitrobenzene (1,000 mg l⁻¹). Moreover, In the presence of salinity (≤5% NaCl, w/v), phenol (≤150 mg l⁻¹) and aniline (≤50 mg l⁻¹), respectively, degradation efficiency of nitrobenzene by PFIC reached over 95%. Even in the presence of both 100 mg l⁻¹ phenol and 50 mg l⁻¹ aniline, over 75% nitrobenzene was removed by PFIC in 36 h. Therefore, the immobilization of the defined consortium in polyurethane foam has application potential for removing nitrobenzene in industrial wastewater treatment system.     

Continuous production of manganese peroxidase by Phanerochaete chrysosporium immobilized on polyurethane foam in a pulsed packed-bed bioreactor

The bottleneck of the application of manganese peroxidase (MnP) on an industrial scale in pulp biobleaching or in degradation of hazardous compounds is the lack of an efficient production system. Three main problems arise for the continuous production of MnP during secondary metabolism of Phanerochaete chrysosporium: enzyme production occurs only under specific physiological conditions corresponding to C or N limitation, high O(2) tension, and adequate Mn(+2) concentration; the enzyme that is produced is destabilized by extracellular proteases; and excessive growth of the mycelium blocks effective oxygen transfer. To overcome these drawbacks, continuous production of MnP was optimized by selecting a suitable bioreactor configuration and the environmental and operating conditions affecting both enzyme production and stability. The combination between a proper feed rate and the application of a pulsation in a packed-bed bioreactor permitted the maintenance of continuous secretion of MnP while limiting mycelial growth and avoiding bed clogging. Environmental factors as an Mn(+2) concentration of 5000 muM and high oxygen tension enhanced MnP production. The hydraulics of the bioreactor corresponding to a plug flow model with partial mixing and an operating hydraulic rentention time of 24 h were optimal to achieve stable operating conditions. This policy allowed long operation periods, obtaining higher productivities than the best reported in the literature. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 130-137, 1997.     

Improving the activity and stability of Yarrowia lipolytica lipase Lip2 by immobilization on polyethyleneimine-coated polyurethane foam

In this study, polyurethane foam (PUF) was used for immobilization of Yarrowia lipolytica lipase Lip2 via polyethyleneimine (PEI) coating and glutaraldehyde (GA) coupling. The activity of immobilized lipases was found to depend upon the size of the PEI polymers and the way of GA treatment, with best results obtained for covalent-bind enzyme on glutaraldehyde activated PEI-PUF (MW 70,000 Da), which was 1.7 time greater activity compared to the same enzyme immobilized without PEI and GA. Kinetic analysis shows the hydrolytic activity of both free and immobilized lipases on triolein substrate can be described by Michaelis–Menten model. The K_m for the immobilized and free lipases on PEI-coated PUF was 58.9 and 9.73 mM, respectively. The V_max values of free and immobilized enzymes on PEI-coated PUF were calculated as 102 and 48.6 U/mg enzyme, respectively. Thermal stability for the immobilization preparations was enhanced compared with that for free preparations. At 50 °C, the free enzyme lost most of its initial activity after a 30 min of heat treatment, while the immobilized enzymes showed significant resistance to thermal inactivation (retaining about 70% of its initial activity). Finally, the immobilized lipase was used for the production of lauryl laurate in hexane medium. Lipase immobilization on the PEI support exhibited a significantly improved operational stability in esterification system. After re-use in 30 successive batches, a high ester yield (88%) was maintained. These results indicate that PEI, a polymeric bed, could not only bridge support and immobilized enzymes but also create a favorable micro-environment for lipase. This study provides a simple, efficient protocol for the immobilization of Y. lipolytica lipase Lip2 using PUF as a cheap and effective material.     

Continuous production of lignin-degrading enzymes by Bjerkandera adusta immobilized on polyurethane foam

Continuous production of lignin-degrading enzymes by Bjerkandera adusta immobilized on polyurethane foam gave maximum activities of 220 U lignin peroxidase ml^–1, 150 U manganese peroxidase ml^–1, 50 U laccase ml^–1 and 6.2 U protease ml^–1 at the retention time of 24 h for 60 days. Protease secretion destabilized the produced lignin peroxidase, manganese peroxidase and laccase.     

Optimization of lactic acid production by immobilized <Emphasis Type="Italic">Lactococcus lactis</Emphasis> IO-1

Production of lactic acid from glucose by immobilized cells of Lactococcus lactis IO-1 was investigated using cells that had been immobilized by either entrapment in beads of alginate or encapsulation in microcapsules of alginate membrane. The fermentation process was optimized in shake flasks using the Taguchi method and then further assessed in a production bioreactor. The bioreactor consisted of a packed bed of immobilized cells and its operation involved recycling of the broth through the bed. Both batch and continuous modes of operation of the reactor were investigated. Microencapsulation proved to be the better method of immobilization. For microencapsulated cells at immobilized cell concentration of 5.3 g l⁻¹, the optimal production medium had the following initial concentrations of nutrients (g l⁻¹): glucose 45, yeast extract 10, beef extract 10, peptone 7.5 and calcium chloride 10 at an initial pH of 6.85. Under these conditions, at 37 °C, the volumetric productivity of lactic acid in shake flasks was 1.8 g l⁻¹ h⁻¹. Use of a packed bed of encapsulated cells with recycle of the broth through the bed, increased the volumetric productivity to 4.5 g l⁻¹ h⁻¹. The packed bed could be used in repeated batch runs to produce lactic acid.     

Mass transfer studies on the reduction of Cr(VI) using calcium alginate immobilized Bacillus sp. in packed bed reactor

This study presents the external mass transfer effects on the reduction of hexavalent chromium (Cr(VI)) using calcium alginate immobilized Bacillus sp. in a re-circulated packed bed batch reactor (RPBR). The effect of flow rate on the reduction Cr(VI) was studied. Theoretically calculated rate constants for various flow rates were analyzed using external film diffusion models and compared with experimental values. The external mass transfer coefficients for the bioconversion of Cr(VI) were also investigated. The external mass transfer effect was correlated with a model of the type J_D = K Re⁻⁽¹⁻ⁿ⁾. The model was tested with various K values and the mass transfer correlation J_D = 5.7 Re^−0.70 was found to predict the experimental data accurately. The proposed model would be useful for the design of industrial reactor and scale up.     

An industrial perspective on bioreactor scale-down: what we can learn from combined large-scale bioprocess and model fluid studies

For industrial bioreactor design, operation, control and optimization, the scale-down approach is often advocated to efficiently generate data on a small scale, and effectively apply suggested improvements to the industrial scale. In all cases it is important to ensure that the scale-down conditions are representative of the real large-scale bioprocess. Progress is hampered by limited detailed and local information from large-scale bioprocesses. Complementary to real fermentation studies, physical aspects of model fluids such as air-water in large bioreactors provide useful information with limited effort and cost. Still, in industrial practice, investments of time, capital and resources often prohibit systematic work, although, in the end, savings obtained in this way are trivial compared to the expenses that result from real process disturbances, batch failures, and non-flyers with loss of business opportunity. Here we try to highlight what can be learned from real large-scale bioprocess in combination with model fluid studies, and to provide suitable computation tools to overcome data restrictions. Focus is on a specific well-documented case for a 30-m(3) bioreactor. Areas for further research from an industrial perspective are also indicated.     

Immobilization of laccase from the white rot fungus Coriolopsis polyzona and use of the immobilized biocatalyst for the continuous elimination of endocrine disrupting chemicals

Laccase from the white rot fungus strain Coriolopsis polyzona was immobilized covalently on the diatomaceous earth support Celite^® R-633 using different strategies. A first methodology involved the sequential activation of the support surface with γ-aminopropyltriethoxysilane followed by the reaction of the functionalized surface with glutaraldehyde (GLU) or glyoxal (GLY) and the immobilization of laccase on the activated surface. Another strategy tested the simultaneous internal cross-linking of the protein with GLU or GLY and the immobilization of the laccase on the silanized surface. Finally, these two strategies were modified to test the impact of the concomitant addition of bovine serum albumin (BSA) as a stabilizing agent during the immobilization steps. The highest laccase activity and the greatest degree of activity recovery (tested using 2,2′-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) as the substrate) were achieved by the sequential immobilization procedure using GLU as the cross-linking agent. The solid catalysts featuring internal cross-linking of the protein showed significantly higher stability against several denaturants. The Michaelis–Menten kinetic parameters with respect to ABTS revealed a higher affinity for this substrate in the case of the sequential procedure compared to the simultaneous approach. The biocatalyst formed using GLU in the sequential procedure was applied in a packed bed reactor for the continuous treatment of 5 mg l⁻¹ solutions of the endocrine disrupting chemicals (EDCs) nonylphenol (NP), bisphenol A (BPA) and triclosan (TCS) through repeated batch treatments. All of these EDCs could be eliminated at a contact time of less than 200 min by using, respectively, 3.75 units (U) of laccase activity for BPA and TCS and 1.88 U for NP. These performances of elimination were maintained over five consecutive treatment cycles using the same biocatalyst. This system could also remove these EDCs from 100 mg l⁻¹ solutions. The Michaelis–Menten kinetic parameters with respect to these chemicals showed a decreasing affinity of the solid biocatalyst for NP, TCS and BPA in that order.     

Kinetics of biodegradation of p-xylene and naphthalene and oxygen transfer in a novel airlift immobilized bioreactor

The scope of this study included the biodegradation performance and the rate of oxygen transfer in a pilot-scale immobilized soil bioreactor system (ISBR) of 10-L working volume. The ISBR was inoculated with an acclimatized population of contaminant degrading microorganisms. Immobilization of microorganisms on a non-woven polyester textile developed the active biofilm, thereby obtaining biodegradation rates of 81 mg/L x h and 40 mg/L x h for p-xylene and naphthalene, respectively. Monod kinetic model was found to be suitable to correlate the experimental data obtained during the course of batch and continuous operations. Oxygen uptake and transfer rates were determined during the batch biodegradation process. The dynamic gassing-out method was used to determine the oxygen uptake rate (OUR) and volumetric oxygen mass transfer, K(L) a. The maximum volumetric OUR of 255 mg O(2)/L x h occurred approximately at 720-722 h after inoculation, when the dry weight of biomass concentration was 0.67 g/L.     

Removal and recovery of uranium from aqueous solutions by Ca-alginate immobilized Trichoderma harzianum

The ability of Ca-alginate immobilized Trichoderma harzianum has been explored for removal and recovery of uranium from aqueous streams. Ca-alginate as polymeric support was selected after screening different matrices. Immobilization of Trichoderma harzianum to Ca-alginate improved the stability as well as uranium biosorption capacity of biosorbent at 28 ± 2 °C and 200 rpm. The suitability of packed bed column operations was illustrated by obtaining break through curves at different bed heights, flow rates and inlet uranium concentrations. The adsorption column containing 1.5 g dry weight of immobilized material has purified 8.5 L of bacterial leach liquor (58 mg/L U) before break through occurred and the biosorbent became saturated after 25 L of influent. Sorbed uranium was recovered in 200 ml of 0.1 N HCl resulting in 98.1–99.3% elution by 0.1 N HCl, which regenerated the biosorbent facilitating the sorption–desorption cycles for better economic feasibility without any significant alteration in sorption capacity/elution efficiency.     

The impact of reduced vacuolar invertase activity on the photosynthetic and carbohydrate metabolism of tomato

The impact of reduced vacuolar invertase activity on photosynthetic and carbohydrate metabolism was examined in tomato (Solanum lycopersicon L.). The introduction of a co-suppression construct (derived from tomato vacuolar invertase cDNA) produced plants containing a range of vacuolar invertase activities. In the leaves of most transgenic plants from line INV-B, vacuolar invertase activity was below the level of detection, whereas leaves from line INV-A and untransformed wild-type plants showed considerable variation. Apoplasmic invertase activity was not affected by the co-suppression construct. It has been suggested that, in leaves, vacuolar invertase activity regulates sucrose content and its availability for export, such that in plants with high vacuolar invertase activity a futile cycle of sucrose synthesis and degradation takes place. In INV-B plants with no detectable leaf vacuolar invertase activity, sucrose accumulated to much higher levels than in wild-type plants, and hexoses were barely detectable. There was a clear threshold relationship between invertase activity and sucrose content, and a linear relationship with hexose content. From these data the following conclusions can be drawn. (i) In INV-B plants sucrose enters the vacuole where it accumulates as hydrolysis cannot take place. (ii) There was not an excess of vacuolar invertase activity in the vacuole; the rate of sucrose hydrolysis depended upon the concentration of the enzyme. (iii) The rate of import of sucrose into the vacuole is also important in determining the rate of sucrose hydrolysis. The starch content of leaves was not significantly different in any of the plants examined. In tomato plants grown at high irradiance there was no impact of vacuolar invertase activity on the rate of photosynthesis or growth. The impact of the cosuppression construct on root vacuolar invertase activity and carbohydrate metabolism was less marked.Abbreviations CaMV Cauliflower Mosaic Virus - WT wild type     

Micropropagation and bioreactor studies of the medicinally important plant Lessertia (Sutherlandia) frutescens L.

This study describes a protocol for rapid and efficient in vitro propagation of Lessertia frutescens (cancer bush), a medicinally important plant species native to southern Africa. Single node explants were grown in various culture regimes of MS medium containing 30 g/l sucrose supplemented with various concentrations of cytokinins and auxins and solidified with 8 g/l agar. These were (a) 2.22, 4.44, 13.32 and 22.19 µM BA; 2.32, 4.65, 13.95 and 23.23 µM K and 0.45, 2.27, 4.54 and 13.62 µM TDZ (b) a combination of 2.22 µM BA with 0.57, 2.85, 5.71 and 11.42 µM IAA, 0.49, 2.46, 4.9 and 9.8 µM IBA or 0.54, 2.69, 5.37 and 10.74 µM NAA and (c) different media types viz. MS, SH basal salt medium and WPM at 1, ½ and ¼ salt strength which were each supplemented with 2.22 µM BA and 0.54 µM NAA. Single node explants were also grown in MS liquid medium supplemented with 2.22 µM BA and 0.54 µM NAA in temporary and continuous immersion bioreactors. Maximum number of shoots (12.9) per single node explant was obtained in the temporary immersion bioreactor but 50% of these shoots showed symptoms of hyperhydricity. In solid culture the best shoot multiplication response (10 shoots) was obtained in full strength MS. Roots were induced using shoot tips cultured in ½ MS solid medium supplemented with various concentrations of IBA or NAA. The highest rooting percentage (78%) was achieved in 19.6 µM IBA. Rooted plantlets were cultured in a mixture of perlite and vermiculite (1:1; v/v) and successfully acclimatized in a growth chamber with an 85% survival rate.     

Synthesis of 2-ethyl hexanol fatty acid esters in a packed bed bioreactor using a lipase immobilized on a textile membrane

An enzymatic process using a packed bed bioreactor with recirculation was developed for the scale-up synthesis of 2-ethylhexyl palmitate with a lipase from Candida sp. 99–125 immobilized on a fabric membrane by natural attachment to the membrane surface. Esterification was effectively performed by circulating the reaction mixture between a packed bed column and a substrate container. A maximum esterification yield of 98% was obtained. Adding molecular sieves and drying the immobilized lipase both decreased the water content at the reactor outlet and around the enzyme, which led to an increase in the rate of esterification. The long-term stability of the reactor was tested by continuing the reaction for 30 batches (over 300 h) with an average esterification yield of about 95%. This immobilized lipase bioreactor is scalable and is thus suitable for industrial production of 2-ethylhexyl palmitate.     

Influence of phenolics on the sensitivity of free and immobilized bioluminescent Acinetobacter bacterium

In this work, the constructed bioluminescent Acinetobacter strain DF4/PUTK2 was employed to assess the toxicity of phenolic compounds and the 5 min EC50 values were calculated. The results of the DF4/PUTK2 assay were further evaluated by comparing with the results of the Vibrio fischeri luminescence inhibition assay. To develop a bioassay system appropriate to be used in continuous toxicity testing, strain DF4/PUTK2 was subjected for immobilization in microtiter plates into the matrices Ca-alginate, polyacrylamide, agar and agarose. After a choice of materials was tried, Ca-alginate was selected as the most suitable candidate material. Because, it could be stored at least 8 weeks at 4 °C, during which the ability of the bioreporter DF4/PUTK2 to detect the toxicity of phenolics was maintained. However, the stability of the bioluminescence for DF4/PUTK2 cells immobilized into agarose and agar was significantly less than that of cells stored in alginate suspensions. This study recommended that luxCDABE-marked Acinetobacter strain DF4/PUTK2 could be employed to assay the ecotoxicity of environmental samples contaminated with phenols. The host strain of the bioreporter DF4/PUTK2 is Acinetobacter strain DF4. It is known that members of the genus Acinetobacter are widespread in nature and also involved in biodegradation, leaching and removal of several organic and inorganic man-made hazardous wastes.     

Kinetics of nutrient utilization and photosynthetic enzyme activities during floral versus vegetative differentiation of Spathiphyllum in air-lift bioreactor cultures

The present study reports on the kinetics of nutrient utilization during in vitro flowering of Spathiphyllum in air-lift bioreactor cultures. Levels of electrical conductivity (EC), anions and cations, pH, ethylene, sugar content and photosynthetic enzymes were determined in bioreactor cultures of both flowering (FPs) and non-flowering (NFPs) plantlets over a growth period of 12 weeks. A decrease in ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity with a corresponding increase in phosphoenolpyruvate carboxylase (PEPcase) activity occurred during floral induction of Spathiphyllum in vitro. Sucrose concentration decreased significantly in FPs, while no changes in glucose, fructose and total sugars were observed in both FPs and NFPs up to 8 weeks of culture. There were significant variations in mineral nutrient utilization between FP and NFP cultures. These results provide an insight to the physiological processes involved in inflorescence formation in Spathiphyllum.     

Applications of laccases and tyrosinases (phenoloxidases) immobilized on different supports: a review

This review summarizes all the research efforts that have been spent to immobilize laccase and tyrosinase for various applications, including synthetic and analytical purposes, bioremediation, wastewater treatment, and must and wine stabilization. All immobilization procedures used in these areas are discussed. Considerations on the efficacy of immobilized copper oxidases and products, in addition to their kinetic parameters are also discussed. The available data indicate that the immobilization of laccase into cationic polymer cross-linked with epichlorohydrin appears to be a promising procedure for industrial applications. The development of laccase and tyrosinase-based biosensors to monitor a wide range of compounds appears to be at a mature stage of technology.     

Enzymatic conversion of sunflower oil to biodiesel in a solvent-free system: Process optimization and the immobilized system stability

The feasibility of using the commercial immobilized lipase from Candida antarctica (Novozyme 435) to synthesize biodiesel from sunflower oil in a solvent-free system has been proved. Using methanol as an acyl acceptor and the response surface methodology as an optimization technique, the optimal conditions for the transesterification has been found to be: 45 ^oC, 3% of enzyme based on oil weight, 3:1 methanol to oil molar ratio and with no added water in the system. Under these conditions, >99% of oil conversion to fatty acid methyl ester (FAME) has been achieved after 50 h of reaction, but the activity of the immobilized lipase decreased markedly over the course of repeated runs. In order to improve the enzyme stability, several alternative acyl acceptors have been tested for biodiesel production under solvent-free conditions. The use of methyl acetate seems to be of great interest, resulting in high FAME yield (95.65%) and increasing the half-life of the immobilized lipase by about 20.1 times as compared to methanol. The reaction has also been verified in the industrially feasible reaction system including both a batch stirred tank reactor and a packed bed reactor. Although satisfactory performance in the batch stirred tank reactor has been achieved, the kinetics in a packed bed reactor system seems to have a slightly better profile (93.6 ± 3.75% FAME yield after 8–10 h), corresponding to the volumetric productivity of 48.5 g/(dm³ h). The packed bed reactor has operated for up to 72 h with almost no loss in productivity, implying that the proposed process and the immobilized system could provide a promising solution for the biodiesel synthesis at the industrial scale.     

Production of L(+)-lactic acid from glucose and starch by immobilized cells of Rhizopus oryzae in a rotating fibrous bed bioreactor

A rotating fibrous-bed bioreactor (RFB) was developed for fermentation to produce L(+)-lactic acid from glucose and cornstarch by Rhizopus oryzae. Fungal mycelia were immobilized on cotton cloth in the RFB for a prolonged period to study the fermentation kinetics and process stability. The pH and dissolved oxygen concentration (DO) were found to have significant effects on lactic acid productivity and yield, with pH 6 and 90% DO being the optimal conditions. A high lactic acid yield of 90% (w/w) and productivity of 2.5 g/L.h (467 g/h.m(2)) was obtained from glucose in fed-batch fermentation. When cornstarch was used as the substrate, the lactic acid yield was close to 100% (w/w) and the productivity was 1.65 g/L.h (300 g/h.m(2)). The highest concentration of lactic acid achieved in these fed-batch fermentations was 127 g/L. The immobilized-cells fermentation in the RFB gave a virtually cell-free fermentation broth and provided many advantages over conventional fermentation processes, especially those with freely suspended fungal cells. Without immobilization with the cotton cloth, mycelia grew everywhere in the fermentor and caused serious problems in reactor control and operation and consequently the fermentation was poor in lactic acid production. Oxygen transfer in the RFB was also studied and the volumetric oxygen transfer coefficients under various aeration and agitation conditions were determined and then used to estimate the oxygen transfer rate and uptake rate during the fermentation. The results showed that the oxygen uptake rate increased with increasing DO, indicating that oxygen transfer was limited by the diffusion inside the mycelial layer.     

Effect of culture medium and illumination on the acid invertase activity in callus of Medicago strasseri

The different acid invertase activity (total, soluble, wall-bound and extracellular) in calli induced on explants (cotyledon, petiole, hypocotyl and leaf) originated from Medicago strasseri seedlings were evaluated. In cultures subjected to 16 h photoperiod, the highest total, soluble and extracellular activities were found in calli from leaves cultured in medium 12 (MS with 0.01 mg·dm⁻³ (0.045 μM) of TDZ), elevated amounts of total and wall-bound invertase being found in calli induced on petioles in 12G medium (MS with 0.01 mg·dm⁻³ (0.045 μM) TDZ and 3.104 mg·dm⁻³ glycerol). In cultures maintained in darkness, the activity detected was lower than that observed in cultures under light conditions. The highest amounts of enzyme was bound in calli cultured on medium 12 (total and extracellular invertase) -leaves- and medium 12D (MS with 0.001 mg·dm⁻³ (0.0045 μM) TDZ) (soluble invertase) -using hypocotyls. In general, the different forms of invertase activity studied seem to appear in greatest amounts in calli induced under light conditions using leaves as explant and TDZ as growth regulator.     

Aspergillus niger inulinase immobilized in polyurethane foam and treated in pressurized LPG: A potential catalyst for enzymatic synthesis of fructooligosaccharides

Inulinase from Aspergillus niger was immobilized in polyurethane foam (PU). Immobilized catalyst was treated in pressurized liquefied petroleum gas (LPG) system. This biocatalyst was used in the fructooligosaccharide production using sucrose as substrate in aqueous system. The main objective of this study was to evaluate the reaction yield and productivity by using polyurethane foam as a low-cost support for enzyme immobilization in an alternative processes for fructooligosaccharide production in pressurized LPG system with potential for industrial application. The total FOS concentration obtained were 31% as a result of sucrose concentration reduction, and formation of FOS long chain (GF3 and GF4) from kestose (GF2). FOS concentrations of 5%, 22%, and 3% were obtained for GF2, GF3, and GF4, respectively. The methodology suggested in this research work, enzyme immobilization in a low-cost support, and treatment in LPG, showed potential technology for fructooligosaccharide synthesis.