Synergistic interaction between two bacterial isolates in the degradation of carbofuran

The dominant bacteriaPseudomonas sp. andArthrobacter sp. were isolated from the standing water of carbofuran-retreatedAzolla plot.Arthrobacter sp. hydrolysed carbofuran added to the mineral salts medium as a sole source of carbon and nitrogen while no degradation occurred withPseudomonas sp. Interestingly, when the medium containing carbofuran was inoculated with bothArthrobacter sp. andPseudomonas sp., a synergistic increase in its hydrolysis and subsequent release of CO₂ from the side chain was noticed. This synergistic interaction was better expressed at 25° C than at 35° C. Likewise, related carbamates, carbaryl, bendiocarb and carbosulfan were more rapidly degraded in the combined presence of both bacterial isolates.     

Microbiological degradation of pentane by immobilized cells of <Emphasis Type="Italic">Arthrobacter sp.</Emphasis>

The increasing production of several plastics such as expanded polystyrene, widely used as packaging and building materials, has caused the release of considerable amounts of pentane employed as an expanding agent. Today many microorganisms are used to degrade hydrocarbons in order to minimize contamination caused by several industrial activities. The aim of our work was to identify a suitable microorganism to degrade pentane. We focused our attention on a strain of Arthrobacter sp. which in a shake-flask culture produced 95% degradation of a 10% mixture of pentane in a minimal medium after 42days of incubation at 20°C. Arthrobacter sp. cells were immobilized on a macroporous polystyrene particle matrix that provides a promising novel support for cell immobilization. The method involved culturing cells with the expanded polystyrene in shake-flasks, followed by in situ growth within the column. Scanning electron microscopy analysis showed extensive growth of Arthrobacter sp. on the polymeric surface. The immobilized microorganism was able to actively degrade a 10% mixture of pentane, allowing us to obtain a bioconversion yield of 90% after 36h. Moreover, in repeated-batch operations, immobilized Arthrobacter sp. cells were able to maintain 85–95% pentane degradation during a 2month period. Our results suggest that this type of bioreactor could be used in pentane environmental decontamination.     

Enzymatic splitting of penicillin V for the production of 6-APA using immobilized penicillin V acylase

Penicillin V acylase from Fusarium sp. SKF 235 was immobilized on several cation-exchange resins, of which Amberlite CG-50 was preferred. Maximum activity of the immobilized penicillin V acylase was 250 to 280 IU/g dry beads. The pH and temperature optima of the enzyme shifted from 6.5 to 6.8 and 55°C to 60°C, respectively, as a result of immobilization. However, the K _m for penicillin V remained at 10mm. Parameters for producing 6-aminopenicillanic acid were investigated and the immobilized penicillin V acylase was used for 68 cycles in a stirred tank reactor.     

Polyelectrolyte complex formation mediated immobilization of chitosan-invertase neoglycoconjugate on pectin-coated chitin

Saccharomyces cerevisiae invertase, chemically modified with chitosan, was immobilized on pectin-coated chitin support via polyelectrolyte complex formation. The yield of immobilized enzyme protein was determined as 85% and the immobilized biocatalyst retained 97% of the initial chitosan-invertase activity. The optimum temperature for invertase was increased by 10 °C and its thermostability was enhanced by about 10 °C after immobilization. The immobilized enzyme was stable against incubation in high ionic strength solutions and was 4-fold more resistant to thermal treatment at 65 °C than the native counterpart. The biocatalyst prepared retained 96 and 95% of the original catalytic activity after ten cycles of reuse and 74 h of continuous operational regime in a packed bed reactor, respectively.     

Immobilization of endo-polygalacturonase from Aspergillus ustus on silica gel

Endo-polygalacturonase from Aspergillus ustus when immobilized on to modified silica gel retained 28% of its original activity. The immobilized enzyme could be re-used through 10 cycles of reaction with almost 90% retention of its original activity. It had increased thermostability over its soluble form: the half-life of the soluble enzyme at 40 °C was less than 10 h whereas the immobilized enzyme retained 82% of its activity after 10 h at 40 °C. Similarly, at 50 °C the half-life of the soluble enzyme was 30 min whereas that of the immobilized enzyme was 5 h.     

Ethanol production at 45 ° by an alginate-immobilized,thermotolerant strain of Kluyveromyces marxianus following growth on glucose-containing media

Summary In comparing ethanol production by free and Ca-alginate immobilized cultures of the thermotolerant yeast, Kluyveromyces marxianus IMB3, on glucose-containing media at 45°C, it was found that initial yields produced by the immobilized culture lagged behind those produced by cultures in free suspension. However, in subsequent batch-feed experiments it was demonstrated that the ethanol-producing ability of the immobilized preparation increased with successive feeds, while production by the free suspension reduced significantly.     

Immobilized, thermostable S- and F-forms of the extracellular invertase from Candida utilis can hydrolyse sucrose up to 100 °C

When grown on a sucrose-containing medium, Candida utilis synthesizes and secretes two invertases: one of molecular size of 280 kDa (the S-form – Slow-migrating) and a new form of Mr of 62 kDa (the F-form – Fast-migrating). Prior to immobilization, purification of S- and F-forms of invertase increased the immobilization yield to 89–100%, in comparison with that of crude invertase preparation (52%). The immobilized purified S- and F-form of invertase remained partially active after 15 min at 100 °C; the F-form retained almost 30% of its maximum activity. The immobilized S-form or F-form of invertase almost completely inverted (95% hydrolysis) 60% (w/v) sucrose over 5 h continuous reaction at 80 °C. Moreover, at 90 °C the immobilized F-form hydrolysed 70% of 60% (w/v) sucrose over 5 h, while the capability of the immobilized S-form of inverting sucrose over 5 h reaction decreased from 80% to 45%.     

The catalytic efficiency of trehalose-6-phosphate synthase is effected by the N-loop at low temperatures

The enzyme OtsA (trehalose-6-phosphate synthase) is ubiquitous in both prokaryotic and eukaryotic organisms, where it plays a critical role in stress resistance and glucose metabolism. Here, we cloned the otsA gene from Arthrobacter sp. Cjts, and expressed and then purified the recombinant proteins. Enzyme activity analysis indicated that the high catalytic efficiency of OtsA from Arthrobacter sp. Cjts resulted from the high affinity of the enzyme for uridine 5′-diphosphoglucose (UDP-Glc) at low temperatures. We also confirmed that the N-loop sequence of OtsA has a large effect on its affinity for UDP-Glc. Sequence analysis indicated that the flexibility of the N-loop may be directly related to the catalytic efficiency of OtsA at low temperatures.     

Recycling of textile bleaching effluents for dyeing using immobilized catalase

Catalase was immobilized on alumina carrier and crosslinked with glutaraldehyde. Storing stability, temperature and pH profiles of enzyme activity were studied in a column reactor with recirculation and in a batch stirred-tank reactor. The immobilized enzyme retained 44% of its activity at pH 11, 30 °C and 90% at 80 °C, pH 7. The half-life time of the immobilized catalase was increased to 2 h at pH 12, and 60 °C. Acceptable results were achieved when the residual water from the washing process of H₂O₂-bleached fabrics was treated with the immobilized enzyme and then reused for dyeing.     

Grape skins as a natural support for yeast immobilization

Grape skins were used to immobilize Saccharomyces cerevisiae. In repeated batch fermentations of grape by immobilized and free cells, the maximum specific rate of alcohol production on glucose decreased from 7.98 h^–1 at 25 °C to 0.7 h^–1 at 5 °C. The rate was approximately twice as high as that on fructose. The rates for free cells were very low. The maximum alcohol yield (0.45 g g^–1) was obtained at 5 °C when the immobilized biocatalyst was used.     

Microbial degradation of pentachlorophenol

Pentachlorophenol (PCP) was the most prevalent wood preservative for many years worldwide. Its widespread use had led to contamination of various environments. Traditional methods of PCP clean-up include storage in land-fill sites, incineration and abiotic degradation processes such as photodecomposition. Some aerobic and anaerobic microorganisms can degrade PCP under a variety of conditions. Axenic bacterial cultures, Flavobacterium sp., Rhodococcus sp., Arthrobacter sp., Pseudomonas sp., Sphingomonas sp., and Mycobacterium sp., and fungal cultures, Phanerochaete sp. and Trametes sp. exhibit varying rates and extent of PCP degradation. This paper provides some general information on properties of PCP and reviews the influence of nutrient amendment, temperature and pH on PCP degradation by various aerobic and anaerobic microorganisms. Where information is available, proposed degradation pathways, intermediates and enzymes are reviewed.     

Enzymatic digestion of spent yeast cells for nutrient recycling in inulase production

Summary An enzyme complex capable of lysing yeast cells was produced byArthrobacter sp. in a medium containing live cells ofKluyveromyces fragilis as the sole source of nutrients. The enzyme complex caused a 90% reduction in the optical density of viable yeast cells in 6 h at 25°C. This yeast cell hydrolysate can be used as a source of nitrogen, vitamins and minerals for subsequent growth of yeast cells (8.3 mg/ml) and further production of inulase (167 U/ml) representing 88 and 87% yield respectively, compared to cells grown on a standard yeast extract (1%) and sucrose (2%) medium.     

Immobilization of alkaline protease from Conidiobolus macrosporus for reuse and improved thermal stability

Alkaline protease from Conidiobolus macrosporus was immobilized on polyamide using glutaraldehyde as a bifunctional agent. The immobilized enzyme was optimally active at a higher temperature of 50°C than the free enzyme (40°C ) and showed a ten-fold increased thermostability at 60°C compared to that of the free enzyme. The efficiency of immobilization was 58% under the optimal conditions of pH and temperature. There was a 14-fold decrease in the K _m of immobilized enzyme compared to the free enzyme. The immobilized enzyme was fully active even after twenty-two cycles of repeated use. It retained 80% activity at 50°C in presence of 8 M urea exhibiting its stability to the denaturant and was compatible with several commercial detergents.     

Effects of pre-storage conditions on storage of in vitro cultures of Nephrolepis exaltata (L.) Schott and Cordyline fruticosa (L.) A. Chev.

In vitro cultures of Nephrolepis exaltata and Cordyline fruticosa were stored at 5°, 9° or 13°C, at a low irradiance (3–5 mol m^–2 s^–1) or in darkness. Prior to storage the cultures were subjected to 18°, 21°, 24° or 27°C and 15, 30 or 45 mol m^–2 s^–1 in a factorial combination.The optimal storage conditions for Nephrolepis were 9°C in complete darkness. These cultures were still transferable to a peat/perlite mixture at the end of the experimental period of 36 months.The optimal storage conditions for Cordyline were 13°C and a low light level (±3–5 mol m^-2 s^-1). When the pre-storage conditions were normal growth room conditions (24°C and 30 mol m^-2 s^-1), in vitro cultures could be stored for 18 months. With the most favourable pre-storage treatment (18°C and 15 mol m^-2 s^-1) some cultures still had green shoots after 36 months of storage, but did not survive transfer to peat/perlite.Pre-conditioning before storage was most favourable for Nephrolepis, and not that important, but still favourable, for Cordyline. There was an interaction between pre-storage temperature and pre-storage irradiance. For both species a high irradiance level was less favourable than a low irradiance level when combined with high growth room temperatures.Abbreviations BA 6-benzyladenine - IAA indole-3-acetic acid - NOA 2-naphthoxyacetic acid     

Characterization of <Emphasis Type="Italic">Mucor miehei</Emphasis> lipase immobilized on polysiloxane-polyvinyl alcohol magnetic particles

Mucor miehei lipase was immobilized on magnetic polysiloxane-polyvinyl alcohol particles by covalent binding. The resulting immobilized biocatalyst was recycled by seven assays, with a retained activity around 10% of its initial activity. K_m and V_max were respectively 228.3 M and 36.1 U mg of protein^–1 for immobilized enzyme. Whereas the optimum temperature remained the same for both soluble and immobilized lipase (45 °C), there was a shift in pH profiles after immobilization. Optimum pH for the immobilized lipase was 8.0. Immobilized enzyme showed to be more resistant than soluble lipase when assays were performed out of the optimum temperature or pH.     

Silanized palygorskite for lipase immobilization

Lipase from Candida lipolytica has been immobilized on 3-aminopropyltriethoxysilane-modified palygorskite support. Scanning electron micrographs proved the covalently immobilization of C. lipolytica lipase on the palygorskite support through glutaraldehyde. Using an optimized immobilization protocol, a high activity of 3300 U/g immobilized lipase was obtained. Immobilized lipase retained activity over wider ranges of temperature and pH than those of the free enzyme. The optimum pH of the immobilized lipase was at pH 7.0–8.0, while the optimum pH of free lipase was at 7.0. The retained activity of the immobilized enzyme was improved both at lower and higher pH in comparison to the free enzyme. The immobilized enzyme retained more than 70% activity at 40 °C, while the free enzyme retained only 30% activity. The immobilization stabilized the enzyme with 81% retention of activity after 10 weeks at 30 °C whereas most of the free enzyme was inactive after a week. The immobilized enzyme retains high activity after eight cycles. The kinetic constants of the immobilized and free lipase were also determined. The K_m and V_max values of immobilized lipase were 0.0117 mg/ml and 4.51 μmol/(mg min), respectively.     

Investigation of the synthesis of xylose/glucose isomerases in sixArthrobacter sp. strains

The substrate specificity of isomerases produced by six strains ofArthrobacter sp. was studied. The role of utilizable carbon sources in controlling enzyme biosynthesis was established. All of the strains studied were found to produce xylose isomerases efficiently, converting D-xylose into D-xylulose and D-glucose into D-fructose. All but A.ureafaciens B-6 strains showed low activity toward D-ribose,Arthrobacter sp. B-5 was slightly active toward L-arabinose, andA. ureafaciens B-6 andArthrobacter sp. B-2239, toward L-rhamnose. InArthrobacter sp. B-5, the synthesis of xylose/glucose isomerase was constitutive (i.e., it was not suppressed by readily metabolizable carbon sources. The synthesis of xylose/glucose isomerase induced by D-xylose inArthrobacter sp. strains B-2239, B-2240, B-2241, and B-2242 and by D-xylose and xylitol inA. ureafaciens B-6 was suppressed by readily metabolizable carbon sources in a concentration-dependent manner. The data obtained suggest that D-xylose and/or its metabolites are involved in the regulation of xylose/glucose isomerase synthesis in theArthrobacter sp. strains B-5, B-2239, B-2240, and B-2241.     

Chemical oxygen demand reduction in a whey fermentation

Summary Preparations of living Pseudomonas denitrificans cells immobilized in alginate gel were used in the denitrification of water. In the presence of an exogenous carbon source the entrapped microorganisms reduced nitrate and nitrite to gaseous products and to achieve complete reduction, carbon to nitrogen ratios of over two were required. The effects on denitrification of particle size and the number of bacteria in the gel were investigated. Apparent K_m values for nitrate and nitrite reduction were calculated for free and immobilized cells. When the immobilized cells were incubated in nutrient media, an increase in reduction rate was observed and this was shown to be caused by the growth of cells within the gel particles. Immobilized P. denitrificans cells retained 75% of their initial nitrate reduction capacity after 21 days of storage at +4°C. The operational stability of the alginate-immobilized cells was studied both in batch and in a column which was operated continuously. A column (45 g of alginate-cell fibers in 80 ml) denitrified a high nitrate drinking water (100 mg NO₃/l) with a rate of 300 ml of nitrate and nitrite free water/day/g of gel. The half life for nitrate reduction was estimated to be 30 days.     

Production and stabilization of amylase preparations from rice bran solid medium

A low-cost amylase preparation of dried fermented bran was developed from rice bran solid cultures of Aspergillus oryzae supplemented with soya bean flour (SBF) and cassava starch (3:1) and dried at 50 °C for 4 h. Storage stability of preparations at 4 °C or 30 °C was significantly enhanced (P 0.05) by adding SBF or partially hydrolyzed starch (PHS). While amylase preparations without stabilizer retained 59 and 48% of their activity after 12 weeks storage at 4 and 30 °C respectively, the same preparations fortified with SBF (5% w/v) retained 95 and 94% stability respectively, during the same period. PHS at 5% (w/v) also gave a maximum stability of 94 and 91.8% at 4 and 30 °C, respectively. The unstabilized preparation retained only 42% of its activity compared to the stabilized forms, which retained 82–90% activity after 15 min incubation at 100 °C.     

Immobilization of glucose oxidase on Fe<Subscript>3</Subscript>O<Subscript>4</Subscript>/SiO<Subscript>2</Subscript> magnetic nanoparticles

Glucose oxidase (GOD) was covalently immobilized onto Fe₃O₄/SiO₂ magnetic nanoparticles (FSMNs) using glutaraldehyde (GA). Optimal immobilization was at pH 6 with 3-aminopropyltriethoxysilane at 2% (v/v), GA at 3% (v/v) and 0.143 g GOD per g carrier. The activity of immobilized GOD was 4,570 U/g at pH 7 and 50°C. The immobilized GOD retained 80% of its initial activity after 6 h at 45°C while free enzyme retained only 20% activity. The immobilized GOD maintained 60% of its initial activity after 6 cycles of repeated use and retained 75% of its initial activity after 1 month at 4°C whereas free enzymes retained 62% of its activity.