Biodegradation of methyl tert-butyl ether by a bacterial pure culture.

A bacterial strain, PM1, which is able to utilize methyl tert-butyl ether (MTBE) as its sole carbon and energy source, was isolated from a mixed microbial consortium in a compost biofilter capable of degrading MTBE. Initial linear rates of MTBE degradation by 2 x 10(6) cells ml(-1) were 0.07, 1.17, and 3.56 microg ml(-1) h(-1) for initial concentrations of 5, 50, and 500 microg MTBE ml(-1), respectively. When incubated with 20 microg of uniformly labeled [(14)C]MTBE ml(-1), strain PM1 converted 46% to (14)CO(2) and 19% to (14)C-labeled cells within 120 h. This yield is consistent with the measurement of protein accumulation at different MTBE concentrations from which was estimated a biomass yield of 0.18 mg of cells mg MTBE(-1). Strain PM1 was inoculated into sediment core material collected from a contaminated groundwater plume at Port Hueneme, California, in which there was no evidence of MTBE degradation. Strain PM1 readily degraded 20 microg of MTBE ml(-1) added to the core material. The rate of MTBE removal increased with additional inputs of 20 microg of MTBE ml(-1). These results suggest that PM1 has potential for use in the remediation of MTBE-contaminated environments.     

Detection and quantification of methyl tert-butyl ether-degrading strain PM1 by real-time TaqMan PCR.

The fuel oxygenate methyl tert-butyl ether (MTBE), a widely distributed groundwater contaminant, shows potential for treatment by in situ bioremediation. The bacterial strain PM1 rapidly mineralizes and grows on MTBE in laboratory cultures and can degrade the contaminant when inoculated into groundwater or soil microcosms. We applied the TaqMan quantitative PCR method to detect and quantify strain PM1 in laboratory and field samples. Specific primers and probes were designed for the 16S ribosomal DNA region, and specificity of the primers was confirmed with DNA from 15 related bacterial strains. A linear relationship was measured between the threshold fluorescence (C(T)) value and the quantity of PM1 DNA or PM1 cell density. The detection limit for PM1 TaqMan assay was 2 PM1 cells/ml in pure culture or 180 PM1 cells/ml in a mixture of PM1 with Escherichia coli cells. We could measure PM1 densities in solution culture, groundwater, and sediment samples spiked with PM1 as well as in groundwater collected from an MTBE bioaugmentation field study. In a microcosm biodegradation study, increases in the population density of PM1 corresponded to the rate of removal of MTBE.     

Short communication: Methylparathion degradation by Pseudomonas sp. A3 immobilized in sodium alginate beads

An organophosphate-degrading soil isolate of Pseudomonas sp. A3, immobilized at 5% (wet wt/v) cell mass in 3% (w/v) sodium alginate beads, detoxified 99% of 1 mm methylparathion in 48 h. The beads were re-usable for five batches, the sixth batch only giving 73% methylparathion removal.     

Naturally occurring bacteria similar to the methyl tert-butyl ether (MTBE)-degrading strain PM1 are present in MTBE-contaminated groundwater

Methyl tert-butyl ether (MTBE) is a widespread groundwater contaminant that does not respond well to conventional treatment technologies. Growing evidence indicates that microbial communities indigenous to groundwater can degrade MTBE under aerobic and anaerobic conditions. Although pure cultures of microorganisms able to degrade or cometabolize MTBE have been reported, to date the specific organisms responsible for MTBE degradation in various field studies have not be identified. We report that DNA sequences almost identical (99% homology) to those of strain PM1, originally isolated from a biofilter in southern California, are naturally occurring in an MTBE-polluted aquifer in Vandenberg Air Force Base (VAFB), Lompoc, California. Cell densities of native PM1 (measured by TaqMan quantitative PCR) in VAFB groundwater samples ranged from below the detection limit (in anaerobic sites) to 10(3) to 10(4) cells/ml (in oxygen-amended sites). In groundwater from anaerobic or aerobic sites incubated in microcosms spiked with 10 microg of MTBE/liter, densities of native PM1 increased to approximately 10(5) cells/ml. Native PM1 densities also increased during incubation of VAFB sediments during MTBE degradation. In controlled field plots amended with oxygen, artificially increasing the MTBE concentration was followed by an increase in the in situ native PM1 cell density. This is the first reported relationship between in situ MTBE biodegradation and densities of MTBE-degrading bacteria by quantitative molecular methods.     

Novel method for cell immobilization and its application for production of oligosaccharides from sucrose

AIMS: The purpose of the present investigation was to develop a novel method for cell immobilization. METHODS AND RESULTS: Aureobasidium pullulans cells were mixed with an alginate solution, and the mixture was extruded to form small gel beads as hydrated-immobilized cells. The beads were then placed at -15 degrees C for 6-24 h to induce freeze-dehydration. The freeze-dehydration resulted in shrinkage of beads as a result of water removal reducing bead volume by 82% and bead weight by 85%. The dehydrated beads were successfully used for the production of fructo-oligosaccharides in a model reactor system. CONCLUSIONS: Dehydrated beads may provide some commercial advantages over conventional immobilized cells. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that bioreactor performance can be improved up to two times by the use of the dehydrated beads.     

Detection and Quantification of Methyl tert-Butyl Ether-Degrading Strain PM1 by Real-Time TaqMan PCR

The fuel oxygenate methyl tert-butyl ether (MTBE), a widely distributed groundwater contaminant, shows potential for treatment by in situ bioremediation. The bacterial strain PM1 rapidly mineralizes and grows on MTBE in laboratory cultures and can degrade the contaminant when inoculated into groundwater or soil microcosms. We applied the TaqMan quantitative PCR method to detect and quantify strain PM1 in laboratory and field samples. Specific primers and probes were designed for the 16S ribosomal DNA region, and specificity of the primers was confirmed with DNA from 15 related bacterial strains. A linear relationship was measured between the threshold fluorescence (C_T) value and the quantity of PM1 DNA or PM1 cell density. The detection limit for PM1 TaqMan assay was 2 PM1 cells/ml in pure culture or 180 PM1 cells/ml in a mixture of PM1 with Escherichia coli cells. We could measure PM1 densities in solution culture, groundwater, and sediment samples spiked with PM1 as well as in groundwater collected from an MTBE bioaugmentation field study. In a microcosm biodegradation study, increases in the population density of PM1 corresponded to the rate of removal of MTBE.     

Naturally Occurring Bacteria Similar to the Methyl tert-Butyl Ether (MTBE)-Degrading Strain PM1 Are Present in MTBE-Contaminated Groundwater

Methyl tert-butyl ether (MTBE) is a widespread groundwater contaminant that does not respond well to conventional treatment technologies. Growing evidence indicates that microbial communities indigenous to groundwater can degrade MTBE under aerobic and anaerobic conditions. Although pure cultures of microorganisms able to degrade or cometabolize MTBE have been reported, to date the specific organisms responsible for MTBE degradation in various field studies have not be identified. We report that DNA sequences almost identical (99% homology) to those of strain PM1, originally isolated from a biofilter in southern California, are naturally occurring in an MTBE-polluted aquifer in Vandenberg Air Force Base (VAFB), Lompoc, California. Cell densities of native PM1 (measured by TaqMan quantitative PCR) in VAFB groundwater samples ranged from below the detection limit (in anaerobic sites) to 10³ to 10⁴ cells/ml (in oxygen-amended sites). In groundwater from anaerobic or aerobic sites incubated in microcosms spiked with 10 μg of MTBE/liter, densities of native PM1 increased to approximately 10⁵ cells/ml. Native PM1 densities also increased during incubation of VAFB sediments during MTBE degradation. In controlled field plots amended with oxygen, artificially increasing the MTBE concentration was followed by an increase in the in situ native PM1 cell density. This is the first reported relationship between in situ MTBE biodegradation and densities of MTBE-degrading bacteria by quantitative molecular methods.     

固定化乳酸乳球菌连续生产Nisin的研究

以海藻酸钙为材料 ,固定乳酸乳球菌 (Lactococcuslactissubsp .lactis)SM5 2 6 ,研究不同条件对Nisin合成的影响。结果表明 ,利用 2 %海藻酸钠在 1 0mmol LCaCl2 条件下 ,得到的固定化细胞颗粒稳定性较好 ,可维持 90h无破裂 ;在发酵过程中SYS3培养基中的无机盐成分尤其磷酸盐对固定化颗粒有破坏作用 ;用mSYS3培养基代替SYS3 ,通过 72h三批次循环的半连续培养 ,Nisin活性为 85 0IU mL ,无明显的细胞渗漏现象。连续化生产 70h ,Nisin活性达 1 1 5 0IU mL ,相当于游离细胞的发酵水平。     

Phytate degradation by immobilized<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> phytase in soybean-curd whey

Saccharomyces cerevisiae CY phytase-producing cells were immobilized in calcium alginate beads and used for the degradation of phylate. The maximum activity and immobilization yield of the immobilized phytase reached 280 mU/g-bead and 43%, respectively. The optimal pH of the immobilized cell phytase was not different from that of the free cells. However, the optimum temperature for the immobilized phytase was 50°C, which was 10°C higher than that of the free cells; pH and thermal stability were enhanced as a consequence of immobilization. Using the immobilized phytase, phytate was degraded in a stirred tank bioreactor. Phytate degradation, both in a buffer solution and in soybean-curd whey mixture, showed very similar trends. At an enzyme dosage of 93.9 mU/g-phytate, half of the phytate was degraded after 1 h of hydrolysis. The operational stability of the immobilized beads was examined with repeated batchwise operations. Based on 50% conversion of the phytate and five times of reuse of the immobilized beads, the specific degradation (g phytate/g dry cell weight) for the immobilized phytase increased 170% compared to that of the free phytase.     

Biodegradation of Methyl tert-Butyl Ether by a Bacterial Pure Culture

A bacterial strain, PM1, which is able to utilize methyl tert-butyl ether (MTBE) as its sole carbon and energy source, was isolated from a mixed microbial consortium in a compost biofilter capable of degrading MTBE. Initial linear rates of MTBE degradation by 2 × 10⁶ cells ml⁻¹ were 0.07, 1.17, and 3.56 μg ml⁻¹ h⁻¹ for initial concentrations of 5, 50, and 500 μg MTBE ml⁻¹, respectively. When incubated with 20 μg of uniformly labeled [¹⁴C]MTBE ml⁻¹, strain PM1 converted 46% to ¹⁴CO₂ and 19% to ¹⁴C-labeled cells within 120 h. This yield is consistent with the measurement of protein accumulation at different MTBE concentrations from which was estimated a biomass yield of 0.18 mg of cells mg MTBE⁻¹. Strain PM1 was inoculated into sediment core material collected from a contaminated groundwater plume at Port Hueneme, California, in which there was no evidence of MTBE degradation. Strain PM1 readily degraded 20 μg of MTBE ml⁻¹ added to the core material. The rate of MTBE removal increased with additional inputs of 20 μg of MTBE ml⁻¹. These results suggest that PM1 has potential for use in the remediation of MTBE-contaminated environments.     

Immobilization of laccase from Streptomyces psammoticus and its application in phenol removal using packed bed reactor

Laccase was produced from Streptomyces psammoticus under solid-state fermentation. The enzyme was partially purified by ammonium sulphate precipitation and was immobilized in alginate beads by entrapment method. Calcium alginate beads retained 42.5% laccase activity, while copper alginate beads proved a better support for laccase immobilization by retaining 61% of the activity. Phenol and colour removal from a phenol model solution was carried out using immobilized laccase. Batch experiments were performed using packed bed bioreactor, containing immobilized beads. Reusability of the immobilized matrix was studied for up to 8 successive runs, each run with duration of 6 h. The system removed 72% of the colour and 69.9% of total phenolics from the phenol model solution after the initial run. The immobilized system maintained 50% of its efficiency after eight successive runs. The degradation of phenolic compounds by immobilized laccase was evaluated and confirmed by Thin layer chromatography and nuclear magnetic resonance spectroscopy.     

亚栖热菌透性化细胞的耦合固定化研究

将海藻酸盐凝胶包埋法与交联法和聚电解质静电自组装覆膜法相耦合,对含有海藻糖合酶活性的亚栖热菌的透性化细胞进行了固定化研究。结果表明,利用重氮树脂和聚苯乙烯磺酸钠对海藻酸凝胶微球交替覆膜,可以显著提高凝胶微球在磷酸盐缓冲液中的稳定性,以碳二亚胺对固定化细胞进行交联处理则可以提高固定化细胞中海藻糖合酶的热稳定性。透性化细胞经包埋－交联－覆膜耦合固定化后,酶活回收率为32％,最适酶反应pH值由6.5左右升至7.0左右,最适反应温度未变,仍为60℃。所得固定化细胞间歇反应时,催化麦芽糖转化为海藻糖的转化率可达60％,重复使用4次（每次50℃、反应24h）,酶活损失小于20％,转化率可保持在50％以上。     

Trichloroethylene degradation by cells of a methane-utilizing bacterium,Methylocystis sp. M,immobilized in calcium alginate

When Methylocystis sp. M cells were immobilized in calcium alginate, the resulting cell beads showed optimum trichloroethylene (TCE) degradation activity at pH 7.0 and 35°C. In comparison with free cells, the immobilized cells were more stable at low pH, and to some extent, at higher temperatures. Studies on the kinetics and the influence of cell density suggest that oxygen permeation was a rate-limiting step. Investigation of the storage stability and the optimum concentration of dissolved oxygen revealed that the TCE degradability was greater under anaerobic than aerobic conditions. Although a toxic effect caused by TCE was observed, methane seemed to restore activity, suggesting that the development of a two-step reactor system might be advantageous. The finding that the immobilized cells showed TCE degradation activity in actual groundwater suggests that TCE bioremediation could be achieved through the use of bioreactors with such cells.     

Degradation of chloroform by immobilized cells of <Emphasis Type="Italic">Bacillus</Emphasis> sp. in calcium alginate beads

A Bacillus sp., capable of degrading chloroform, was immobilized in calcium alginate. The beads in 20 g alginate l⁻¹ (about 2 × 10⁸ cells/bead) could be re-used nine times for degradation of chloroform at 40 μM. The immobilized cells had a higher range of tolerance (pH 6.5–9 and 20–41°C) than free cells (pH 7–8.5 and 28–32°C). At 5 g alginate l⁻¹, leakage of the cells from the beads was 0.51 mg dry wt ml⁻¹. This species is the first reported Bacillus that can degrade chloroform as the sole carbon source.     

Solubilization of fish proteins using immobilized microbial cells

Cells of Bacillus megaterium, Aeromonas hydrophila, and Pseudomonas marinoglutinosa were immobilized in calcium alginate. The immobilized cells secreted protease when held in fish meat suspension in water. The enzyme synthesis by the entrapped cells was supported by small amounts of soluble nutrients present in the meat. The secreted protease solubilized the fish meat, solubilization being optimum at pH range of 7.5 to 9.5 and at 50 degrees C. Under these conditions immobilized B. megaterium was most efficient giving 30% solubilization of the meat, followed by A. hydrophila (18%), while immobilized P. marinoglutinosa was less effective. The optimum ratio of fish meat to beads was about 4:3 for B. megaterium and A. hydrophila. The beads had a storage life of 30 days at 4 degrees C. The results suggested potential for use of immobilized microbial cells having extracellular protease activity to enhance solubility of waste proteins. A prototype reactor with beads holding assembly was fabricated which could recover the beads from the meat slurry after the treatment.     

Degradation of 4-aminobenzenesulfonate by alginate encapsulated cells of Agrobacterium sp. PNS-1

Studies were carried out on 4-aminobenzenesulfonate (4-ABS) degradation by free and alginate entrapped cells of Agrobacterium sp. PNS-1. Degradation rate in batch reactors with free cells was marginally higher than Ca-encapsulated cells. Comparison of Ca2+ and Ba2+ as gelling agents showed that 4-ABS removal rate was significantly less with Ba-alginate entrapped cells. Specific degradation rates, using linear regression analysis and based on the initial biomass in the beads, varied from 49.7 mg/mg biomass/h to 92.0 mg/mg biomass/h for Ca-alginate encapsulated cells for different initial 4-ABS concentrations ranging from 200 to 800 mg/L. UV spectra of the aliquots drawn at different time intervals from batch reactors did not show accumulation of any intermediate during degradation. Ca-alginate immobilized cells could be repeatedly reused upto five cycles without any loss of activity. Studies with packed bed reactors, operated in a semi-continuous mode, showed that this could be used for 4-ABS degradation.     

Production of thermostable and neutral glucoamylase using immobilized <Emphasis Type="Italic">Thermomucor indicae-seudaticae</Emphasis>

Thermomucor indicae-seudaticae was immobilized in alginate, κ-carrageenan, agarose, agar, polyacrylamide and loofah (Luffa cylindrica) sponge (as such or coated with alginate/starch/Emerson YpSs agar), and used for the production of glucoamylase in submerged fermentation. The mycelium developed from alginate-immobilized sporangiospores secreted higher glucoamylase titres (22.7 U ml⁻¹) than those immobilized in other gel matrices and the freely growing mycelial pellets (18.5 U ml⁻¹). Loofah network provided a good support for mycelial growth, but the enzyme production was lower than that attained with alginate beads. Glucoamylase production increased with inoculum density and the optimum levels were achieved when 40 calcium alginate beads (∼5 × 10⁶ immobilized spores) were used to inoculate 50 ml production medium. The alginate bead inoculum displayed high storage stability at 4°C and produced comparable enzyme titres up to 120 days. The glucoamylase production by hyphae emerged from the immobilized sporangiospores was almost stable over eight batches of repeated fermentation. Scanning electron micrographs of alginate beads, after batch fermentation, revealed extensive mycelial growth inside and around the beads.     

A comparative study of four systems for tertiary wastewater treatment by Scenedesmus bicellularis: New technology for immobilization

Immobilization appears to be one of the best techniques to separate physically micro-algal cells from their culture medium for the purpose of algal tertiary wastewater treatment. High operation costs and other drawbacks of large-scale physico-chemical methods of harvest led to a comparative study of biotreatment systems. Before treatment began, Scenedesmus bicellularis cells were conditioned (starved) under four different sets of conditions: 1) non-immobilized cells with air bubbling (NCA); 2) cells immobilized in alginate beads (CBW) and 3) cells immobilized on alginate screens (CSW), all conditioned in synthetic culture medium depleted in N and P; 4) cells immobilized on alginate screens but conditioned in air at 100% relative humidity (CSA). Starvation was started under a light:dark photoperiod of 16:8 h. Starved cells were then used to treat wastewater for a 2-h period. The performance of each system was evaluated by determination of residual NH₄-N and phosphate ions and by growth (dry weight, total chlorophyll, cell count, protein content). We then tested the capacity of microalgae immobilized on screens to eliminate N and P from a secondary municipal wastewater effluent and examined the influence of temperature and starvation. The quality of treated effluents was improved considerably with the system using CSA or CSW model. For CSA model, the protein content was 22.4 pg cell^-1 compared to 12.9, 9.5, 9.1 pg cell^-1 for NCA, CBW and CSW models, respectively. The CBW and CSW models were efficient for chlorophyll synthesis. The residual ammonium content in natural wastewater after 2 h of treatment with CSA model was 39% at 6±2 °C and reached 100% removal at 18±2 °C. With the first 2 h, the removal of orthophosphate was inferior (53%) at 6±2 °C, but 88 to 100% at 18±2 °C depending on starvation times. Long starvation times (72 or 96 h) caused damage to cells and uptake of nutrients was lower than with 54 h starvation. This work demonstrates that by using immobilization on screens, removal of nutrients from wastewater was higher than with conventional biological tertiary wastewater treatments (free cells or bead-shaped alginate particles).     

Degradation of benzene by a Rhodococcus sp. using immobilized cell systems

The continuous degradation of benzene by a Rhodococcus sp. using free and immobilized cell systems was compared. Cell entrapment in calcium and strontium alginate beads and adhesion on support materials such as glass beads were found to be unsatisfactory. Degradation of benzene by cells immobilized in either ceramic or cellulose carriers proved to be more efficient than its non-immobilized counterpart. A retention time of 36 h was required to effect a 97% degradation of benzene using suspended free cells while cells immobilized on cellulose or ceramic carriers effected 97% degradation at 24 and 18 h, respectively. Recycling of the ceramic carriers was also possible and resulted in an even shorter retention time of 12h to effect a 97% degradation of benzene. Cell adhesion on the support materials was confirmed by scanning electron microscopy.     

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.