Degradation of naphthalene by cells of Pseudomonas sp. strain NGK 1 immobilized in alginate, agar and polyacrylamide

A Pseudomonas sp. strain NGK 1 (NCIM 5120) was immobilized in various matrices, namely, alginate, agar (1.8 × 10¹¹ cfu g⁻¹ beads) and polyacrylamide (1.6 × 10¹¹ cfu g⁻¹ beads). The degradation of naphthalene was studied, by freely suspended cells (4 × 10¹⁰ cfu ml⁻¹) and immobilized cells in batches, with shaken culture and continuous degradation in a packed-bed reactor. Free cells brought about the complete degradation of 25 mmol naphthalene after 3 days of incubation, whereas, a maximum of 30 mmol naphthalene was degraded by the bacteria after 3–4 days of incubation with 50 mmol and 75 mmol naphthalene, and no further degradation was observed even after 15 days of incubation. Alginate-entrapped cells had degraded 25 mmol naphthalene after 3.5 days of incubation, whereas agar- and polyacrylamide-entrapped cells took 2.5 days; 50 mmol naphthalene was completely degraded by the immobilized cells after 6–7 days of incubation. Maximum amounts of 55 mmol, 70 mmol and 67 mmol naphthalene were degraded, from an initial 75 mmol naphthalene, by the alginate-, agar- and polyacrylamide-entrapped cells after 15 days of incubation. When the cell concentrations were doubled, 25 mmol and 50 mmol naphthalene were degraded after 2 and 5.5 days of incubation by the immobilized cells. Complete degradation of 75 mmol naphthalene occurred after 10 days incubation with agar- and polyacrylamide-entrapped␣cells, whereas only 60 mmol naphthalene was degraded by alginate-entrapped cells after 15 days of␣incubation. Further, with 25 mmol naphthalene, alginate-, agar- and polyacrylamide-entrapped cells (1.8 × 10¹¹ cfu g⁻¹ beads) could be reused 18, 12 and 23 times respectively. During continuous degradation in a packed-bed reactor, 80 mmol naphthalene 100 ml⁻¹ h⁻¹ was degraded by alginate- and polyacrylamide-entrapped cells whereas 80 mmol naphthalene 125 ml⁻¹␣h⁻¹ was degraded by agar-entrapped cells. Received: 21 October 1997 / Received revision: 15 January 1998 / Accepted: 18 January 1998     

Biodegradation of Carbofuran phenol by free and immobilized cells of <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> ATCC13883T

The Klebsiella sp. strain ATCC13883T capable of degrading carbofuran phenol (2,3-dihydro-2,2-dimethylbenzofuran-7-ol) has been separated from the soil by enrichment culture technique and immobilized in various, namely polyurethane foam (PUF), polyacrylamide, alginate, agar and alginate-bentonite clay-powdered activated charcoal (PAC). The degradation rates of 20 and 30 mM carbofuran phenol by free and immobilized cells in batch and semi-continuous shaken cultures were compared. The PUF-immobilized cells achieved higher degradation rates in a shorter time than freely suspended cells and the cells immobilized in polyacrylamide, alginate and agar. The PUF- and alginate-bentonite clay-PAC-immobilized cells could be reused for more than 36 cycles, polyacrylamide-entrapped cells for 20 cycles and alginate-bentonite-PAC 28 cycles, without losing any degradation capacity and showed better tolerance to pH, temperature and concentration changes than free cells. These results showed that cells immobilized in modified alginate-bentonite-PAC immobilizers tolerated and completely degraded carbofuran phenol at initial concentrations of 20 and 30 mM and also higher. Such a bacterial strain could be used for bioremediation of environments contaminated with phenolic compounds.     

Degradation of 2-methylnaphthalene by free and immobilized cells of Pseudomonas sp. strain NGK1

A Pseudomonas sp. strain NGK1 (NCIM 5120) capable of utilizing 2-methylnaphthalene (2-MN) was immobilized in various matrices namely, polyurethane foam (PUF), alginate, agar and polyvinyl alcohol (PVA) (1.5 × 10¹² c.f.u. g^–1 beads). The degradation rates of 25 and 50 mM 2-MN by freely suspended cells (2 × 10¹¹ c.f.u. ml^–1) and immobilized cells in batches, semi-continuous with shaken culture and continuous degradation in a packed-bed reactor were compared. The PUF-immobilized cells achieved higher degradation of 25 and 50 mM of 2-MN than freely suspended cells and the cells immobilized in alginate, agar or PVA. The PVA- and PUF-immobilized cells could be reused for more than 30 and 20 cycles respectively, without losing any degradation capacity. The effect of dilution rates on the rate of degradation of 25 and 50 mM 2-MN with freely suspended and immobilized cells were compared in the continuous system. Increase in dilution rate increased the degradation rate only up to 1 h^–1 in free cells with 25 mM 2-MN and no significant increase was observed with 50 mM 2-MN. With immobilized cells, the degradation rate increased with increase in dilution rate up to 1.5 h^–1 for 25 mM and 1 h^–1 for 50 mM 2-MN. These results revealed that the immobilized cell systems are more efficient than freely suspended cells for biodegradation of 2-MN.     

Loofa sponge as a scaffold for the culture of human hepatocyte cell line

Loofa sponge was investigated as a three-dimensional scaffold for stationary and perfusion culture of human hepatoblastoma cell line C3A/HepG2. In stationary culture, C3A/HepG2 cells in loofa cubes showed higher alpha-fetoprotein and albumin secretion rates than those in polyurethane foam (PU). To use loofa cylinders in a packed-bed reactor, immobilization of C3A/HepG2 cells by recirculating medium at 26 mL/min (superficial velocity = 51.7 cm/min) resulted in a cell loading density of 5.15 x 10(7) cells/cm(3)-loofa. This cell loading density is higher than values reported in the literature for packed-bed reactor intended for bioartificial liver. During 9 days of perfusion culture in the reactor, immobilized C3A/HepG2 showed steady synthesis of albumin with an average synthesis rate at 42.2 microg/10(6) cells/day. These experimental results and observations by SEM suggested that loofa sponge is a suitable scaffold for high-density culture of human hepatocyte cell line and the immobilized cells could express high levels of liver-specific functions.     

Biodegradation of Naphthalene by Pseudomonas stutzeri in Marine Environments: Testing Cells Entrapment in Calcium Alginate for Use in Water Detoxification

The biodegradation of naphthalene in sea water by freely suspended and alginate-entrapped cells of Pseudomonas stutzeri 19SMN4 has been investigated in batch cultures. The results showed that immobilized cells can be stored at 4°C for 1 month without loss of viability. The biodegradation was highly affected by the availability of nitrogen and phosphorous, so at 30°C a naphthalene concentration of 25 mM was almost completely degraded (93%) by free cells in 6 days in samples supplemented with these nutrients, whereas only 42% naphthalene was consumed in the nonsupplemented samples. Biodegradation was much slower at 16°C than at 30°C; after 6 days of culture at 30°C, almost all naphthalene was degradated by free and immobilized cells, whereas only 22% and 34% at 16°C, respectively. The degradation rate remained unaffected when the naphthalene concentration was reduced from 25 to 10 mM. Alginate of three different viscosities was used for immobilization of cells. After 7 days of culture, beads formed with 31.4 cP alginate were fragmented, whereas beads formed with 240 and 3600 cP did not display structural changes and afforded the same degradation rate. Beads formed with high-viscosity alginate retained cells more efficiently.     

Degradation of pentachlorophenol by polyurethane-immobilized Flavobacterium cells

Polyurethane-immobilized Flavobacterium cells (ATCC 39723) degraded pentachlorophenol (PCP) at initial concentrations as high as 300 mg liter-1. The reversible binding of PCP to the polyurethane was shown to be important in the protection of the cells from inhibition of PCP degradation. The degradation activity of the bacteria was monitored for 150 days in semicontinuous batch reactors. The degradation rate dropped by about 0.6% per day. PCP was degraded in a continuous-culture bioreactor at a rate of 3.5 to 4 mg g of foam-1 day-1 for 25 days. Electron micrographs of the polyurethane suggested that the cells were entrapped within 50- to 500-microns-diameter pockets in the foam.     

Degradation of pentachlorophenol by polyurethane-immobilized Flavobacterium cells.

Polyurethane-immobilized Flavobacterium cells (ATCC 39723) degraded pentachlorophenol (PCP) at initial concentrations as high as 300 mg liter-1. The reversible binding of PCP to the polyurethane was shown to be important in the protection of the cells from inhibition of PCP degradation. The degradation activity of the bacteria was monitored for 150 days in semicontinuous batch reactors. The degradation rate dropped by about 0.6% per day. PCP was degraded in a continuous-culture bioreactor at a rate of 3.5 to 4 mg g of foam-1 day-1 for 25 days. Electron micrographs of the polyurethane suggested that the cells were entrapped within 50- to 500-microns-diameter pockets in the foam.     

Citric acid production by Aspergillus niger immobilized on polyurethane foam

Summary Citric acid was produced using Aspergillus niger immobilized on polyurethane foam in a bubble column reactor. Most of the adsorbed cells remained on the support and, as a result, high oxygen tension was maintained during the reactor operation. However, uncontrolled growth of the pellets made continuous reactor operation difficult. The citric acid productivity obtained from 15 vol.% foam particles containing immobilized cells was 0.135 g/l per hour. This productivity of immobilized cells was almost the same as that of free cells. The oxygen level dropped to half saturation in 5 days in the immobilized cell culture in contrast to 2 days in the free cell culture.     

Kinetics of p-cresol degradation by an immobilized Pseudomonas sp

A p-cresol (PCR)-degrading Pseudomonas sp. was isolated from creosote-contaminated soil and shown to degrade PCR by conversion to protocatechuate via p-hydroxybenzaldehyde (PBA) and p-hydroxybenzoate (PHB). Cells of the Pseudomonas sp. were immobilized in calcium alginate beads and in polyurethane foam. The relationship between the PCR concentration and the PCR transformation rate was investigated in batch and continuous culture bioreactors. The biodegradation kinetics of PBA and PHB also were investigated. In batch culture reactors, the maximum PCR degradation rate (Vmax) for the alginate-immobilized Pseudomonas sp. cells was 1.5 mg of PCR g of bead-1 h-1 while the saturation constant (Ks) was 0.22 mM. For PHB degradation, the Vmax was 0.62 mg of PHB g of bead-1 h-1 while the Ks was 0.31 mM. For polyurethane-immobilized Pseudomonas sp. cells, the Vmax of PCR degradation was 0.80 mg of PCR g of foam-1 h-1 while the Ks was 0.28 mM. For PHB degradation, the Vmax was 0.21 mg of PHB g of foam-1 h-1 and the Ks was 0.22 mM. In a continuous column alginate bead reactor, the Vmax for PCR transformation was 2.6 mg g of bead-1 h-1 while the Ks was 0.20 mM. The Vmax and Ks for PBA transformation in the presence of PCR were 0.93 mg g of bead-1 h-1 and 0.063 mM, respectively. When PHB alone was added to a reactor, the Vmax was 1.48 mg g of bead-1 h-1 and the Ks was 0.32 mM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of p-cresol degradation by an immobilized Pseudomonas sp.

A p-cresol (PCR)-degrading Pseudomonas sp. was isolated from creosote-contaminated soil and shown to degrade PCR by conversion to protocatechuate via p-hydroxybenzaldehyde (PBA) and p-hydroxybenzoate (PHB). Cells of the Pseudomonas sp. were immobilized in calcium alginate beads and in polyurethane foam. The relationship between the PCR concentration and the PCR transformation rate was investigated in batch and continuous culture bioreactors. The biodegradation kinetics of PBA and PHB also were investigated. In batch culture reactors, the maximum PCR degradation rate (Vmax) for the alginate-immobilized Pseudomonas sp. cells was 1.5 mg of PCR g of bead-1 h-1 while the saturation constant (Ks) was 0.22 mM. For PHB degradation, the Vmax was 0.62 mg of PHB g of bead-1 h-1 while the Ks was 0.31 mM. For polyurethane-immobilized Pseudomonas sp. cells, the Vmax of PCR degradation was 0.80 mg of PCR g of foam-1 h-1 while the Ks was 0.28 mM. For PHB degradation, the Vmax was 0.21 mg of PHB g of foam-1 h-1 and the Ks was 0.22 mM. In a continuous column alginate bead reactor, the Vmax for PCR transformation was 2.6 mg g of bead-1 h-1 while the Ks was 0.20 mM. The Vmax and Ks for PBA transformation in the presence of PCR were 0.93 mg g of bead-1 h-1 and 0.063 mM, respectively. When PHB alone was added to a reactor, the Vmax was 1.48 mg g of bead-1 h-1 and the Ks was 0.32 mM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biodegradation of p-cresol by immobilized cells of Bacillus sp. strain PHN 1

The Bacillus sp. strain PHN 1 capable of degrading p-cresol was immobilized in various matrices namely, polyurethane foam (PUF), polyacrylamide, alginate and agar. The degradation rates of 20 and 40 mM p-cresol by the freely suspended cells and immobilized cells in batches and semi-continuous with shaken cultures were compared. The PUF-immobilized cells achieved higher degradation of 20 and 40 mM p-cresol than freely suspended cells and the cells immobilized in polyacrylamide, alginate and agar. The PUF- immobilized cells could be reused for more than 35 cycles, without losing any degradation capacity and showed more tolerance to pH and temperature changes than free cells. These results revealed that the immobilized cell systems are more efficient than freely suspended cells for degradation of p-cresol.     

Enhanced degradation of 3-nitrobenzoate by immobilized cells of Bacillus flexus strain XJU-4

Nitroaromatic compounds are major chemical pollutants because of their widespread use and toxicity. Bioremediation of such toxic nitroaromatic compounds using microorganisms may provide an effective method for detoxification. Bacillus flexus strain XJU-4, capable of degrading 3-nitrobenzoate, was immobilized in various matrices, namely polyurethane foam (PUF), polyacrylamide, sodium alginate (SA), sodium alginate-polyvinyl alcohol (SA-PVA) and agar. The degradation of 12 and 24 mM 3-nitrobenzoate, by both freely suspended cells and immobilized cells, in batches and fed-batch with shaken cultures were compared. The PUF-immobilized cells achieved higher degradation rates of 12 and 24 mM — nitrobenzoate than freely suspended cells, and the cells immobilized in SA-PVA, polyacrylamide, SA and agar. The PUF-immobilized cells could be reused for more than 21 cycles without losing any degradation capacity. These results revealed the feasibility of using PUF-immobilized cells of B. flexus for the enhanced degradation of — nitrobenzoate.     

对-氯代苯胺(PCA)在填充床生物反应器中的降解作用

以氯代苯胺(PCA)为选择基质,用驯化技术从降解对二氯苯(p-DCB)的富集培养物中得到了以同化PCA为唯一碳源和氮源的混合微生物。将这种固定在填充床反应器中的微生物用于PCA的降解作用研究中。在该反应器里,PCA的生物降解遵循Logistic方程q=q_max/(1+e^α-βU_v).由方程求出了主要的动力学常数,K_s(半速率常数)和q_max(最大比基质降解速率).于PCA降解的同时,释放氯离子到培养基中。在水力停留时间3h, 进水PCA浓度为360mg·L^-1情况下,基质的体积降解率达到125mg·L^-1·h^-1;基质的百分去除率为91%.     

Screening of encapsulated microbial cells for the degradation of inorganic cyanides

Summary Different encapsulation matrices were screened to encapsulate cells ofPseudomonas putida for degradation of inorganic cyanides. Degradation of NaCN by free cells and cells immobilized in agar, alginate or carrageenan matrices was studied. The rate of NaCN degradation was monitored for 120 h by measuring pH, bacterial growth, dissolved and gaseous NH₃ and gaseous CO₂. Alginate-immobilized cells degraded NaCN more efficiently than free cells or agar- or carrageenan-immobilized cells.     

Use of microrespirometry to determine viability of immobilized Burkholderia cepacia G4

Embedding of Burkholderia cepacia G4 cells in a polyurethane-based foam decreased their culturability by more than four orders of magnitude. However, respiration rates of immobilized cells were at least 33-41% of unimmobilized cells. Embedded cells also degraded trichloroethylene. Therefore, respirometry is a more reliable indicator of viability of polyurethane immobilized bacteria than culturing methods.     

Covalent binding of a nerve agent hydrolyzing enzyme within polyurethane foams

A phosphotriesterase preparation, extracted from Escherichia coli DH5alpha cells, was immobilized within a polyurethane foam matrix during polymer synthesis. The enzyme-foam interaction was shown to be covalent and analysis of the hydrolysis of paraoxon in aqueous solution demonstrated that more than 50% of the initial enzyme specific activity was retained after immobilization in the foam. Factors affecting the rate of paraoxon degradation include foam hydrophobicity, the degree of mixing applied to initiate polymerization, and foam pretreatment prior to use in substrate hydrolysis. The storage stability of the foam is significant, with phosphotriesterase-foam activity profiles exhibiting a three month half-life. Foams are currently being developed for biocatalytic air filtering, in which gaseous substrates will be simultaneously adsorbed and degraded by the immobilized enzyme system. (c) 1996 John Wiley & Sons, Inc.     

The use of silica gel prepared by sol-gel method and polyurethane foam as microbial carriers in the continuous degradation of phenol

A mixed microbial culture was immobilized by entrapment into silica gel (SG) and entrapment/ adsorption on polyurethane foam (PU) and ceramic foam. The phenol degradation performance of the SG biocatalyst was studied in a packed-bed reactor (PBR), packed-bed reactor with ceramic foam (PBRC) and fluidized-bed reactor (FBR). In continuous experiments the maximum degradation rate of phenol (q _s ^max) decreased in the order: PBRC (598 mg l⁻¹h⁻¹) > PBR (PU, 471 mg l⁻¹h⁻¹) > PBR (SG, 394 mg l⁻¹h⁻¹) > FBR (PU, 161 mg l⁻¹h⁻¹) > FBR (SG, 91 mg l⁻¹ h⁻¹). The long-term use of the SG biocatalyst in continuous phenol degradation resulted in the formation of a 100–200 μm thick layer with a high cell density on the surface of the gel particles. The abrasion of the surface layer in the FBR contributed to the poor degradation performance of this reactor configuration. Coating the ceramic foam with a layer of cells immobilized in colloidal SiO₂ enhanced the phenol degradation efficiency during the first 3 days of the PBRC operation, in comparison with untreated ceramic packing. Received: 2 December 1999 / Revision received: 2 February 2000 / Accepted: 4 February 2000     

Biodegradation of naphthalene by free and alginate entrapped Pseudomonas sp

Naphthalene degradation by freely suspended and immobilized cells of Pseudomonas sp. isolated from contaminated effluents has been investigated in batch cultures and continuously in a packed bed reactor. Naphthalene concentration was varied from 25 mM to 75 mM, the temperature (30 degrees C) and pH (7.0) were kept constant. The results showed good acclimation of the strain to carbon source and degradation rate was highly affected by initial concentration. Alginate-entrapped cells have given good yields although initial rates were not as high as those encountered with free cells. A first order exponential decay kinetic model was proposed with values of parameters for each initial concentration. A laboratory scale packed-bed bioreactor was designed using parameters calculated above and continuous experiments were realized at different flow rates (100 to 200 ml/h), with different feed concentrations and operating during 30 days. The conversion at low feed concentrations and low flow rates was complete whereas at high flow rates and high concentrations it was less efficient because of diffusional limitations and short residence time.     

Importance of Gram-positive naphthalene-degrading bacteria in oil-contaminated tropical marine sediments

AIMS: The aim of this study was to isolate, characterize and evaluate the importance of naphthalene-degrading bacterial strains from oil-contaminated tropical marine sediments. METHODS AND RESULTS: Three Gram-positive naphthalene-degrading bacteria were isolated from oil-contaminated tropical intertidal marine sediments by direct isolation or enrichment using naphthalene as the sole source of carbon and energy. Bacillus naphthovorans strain MN-003 can also grow on benzene, toluene, xylene and diesel fuel while Micrococcus sp. str. MN-006 can also grow on benzene. Staphylococcus sp. str. MN-005 can only degrade naphthalene and was not able to use the other aromatic hydrocarbons tested. Strain MN-003 possessed the highest maximal specific growth rate with naphthalene as sole carbon source. An enrichment culture fed with naphthalene as sole carbon source exhibited a significant increase in the relative abundances of the three isolates after 21 days of incubation. The three isolates constituted greater than 69% of the culturable naphthalene-degrading microbial community. Strain MN-003 outcompeted and dominated the other two isolates in competition studies involving batch cultures inoculated with equal cell densities of the three isolates and incubated with between 1 and 10 mg l-1 of naphthalene. CONCLUSIONS: Three Gram-positive naphthalene-degrading bacteria were successfully isolated from oil-contaminated tropical marine sediments. Gram-positive bacteria might play an important role in naphthalene degradation in the highly variable environment of oil-contaminated tropical intertidal marine sediments. Among the three isolates, strain MN-003 has the highest maximal specific growth rate when grown on naphthalene, and outgrew the other two isolates in competition experiments. SIGNIFICANCE AND IMPACT OF THE STUDY: This research will aid in the development of bioremediation schemes for oil-contaminated marine environments. Strain MN-003 could potentially be exploited in such schemes.     

Immobilized cyanobacteria as a biofertilizer for rice crops; Intl. Conference on Applied Algology, Knysna, South Africa, April 1996.

N₂-fixing cyanobacteria (Anabaena azollae, symbiont strains) were immobilized in polyurethane foam and ammonia production by the cyanobacteria was investigated in the laboratory and rice field. The cyanobacterial symbiont, A. azollae - MPK-SK-AM-24 showed the highest growth rate and biomass production amongst the 5 isolates examined while A. azollae-AS-DS showed the highest nitrogenase activity followed by A. variabilis - SA₀ (wild type, non-symbiotic). Treatment of the foam-immobilized cyanobacteria with the systemic fungicide Bavistin stimulated nitrogenase activity while inhibiting glutamine synthetase (GS) activity. Free-living A. azollae-MPK-SK-AF-38, A. azollae - MPK-SK-AM-24 and A. azollae-MPK-SK-AM-27 excreted the highest amounts of ammonia into the growth medium; under foam - immobilized conditions the ammonia production increased further. Treatment of the foam - immobilized cyanobacteria with the fungicides Bavistin and Vitavax resulted in ammonia production at significantly higher rates. Rice seedlings (var. ADT 36) grown in the laboratory in conjunction with foam - immobilized A. azollae showed increased growth. A field experiment with paddy rice and foam - immobilized A. azollae strains indicated that the cyanobacteria excreted significant amounts of ammonia into the flood water in the rice fields resulting in increased chlorophyll content of the plants and increased the rice grain and straw yields. A combination of fertilizer nitrogen and inoculation with foam - immobilized cyanobacteria also significantly increased the rice grain and straw yield. Additionally, both A. azollae and A. variabilis were immobilized in sugarcane waste (bagasse), added to rice paddy and resulted in increased rice grain yield. This revised version was published online in September 2006 with corrections to the Cover Date.