Biosorption of Zinc using Bacillus subtilis (MTCC 2423)

Heavy metals such as zinc in untreated industrial effluents cause diseases and disorders in living organisms. They cannot be degraded like organic contaminants and hence have to be removed. Though physical and chemical methods are available for their removal, most of them are not economical and eco-friendly. Hence, a suitable technique is necessary to minimize the deleterious effects of dispersion of heavy metals in ecosystems. Though zinc serves as a micronutrient, it becomes toxic in higher concentrations. Bacteria can be used in the removal of zinc and the process is economical and ecofriendly. Hence, in the present study, we tested zinc removal efficiency of Bacillus subtilis (MTCC 2423) for various concentrations viz. 100, 200, 300, 400 and 500 ppm of zinc in nutrient broth for a period of 10 days. Samples were tested for the zinc level every two days in each concentration and the maximum removal was noticed after six days of treatment. With the increase in zinc concentration, both biomass and zinc removal efficiency showed an increase. Autoclaved cells showed maximum zinc removal when compared with other cell types. Among the other heavy metals tested, iron enhanced the biomass of B. subtilis during zinc treatment and the results are discussed.

Construction and immobilization of recombinant Bacillus subtilis with D-allulose 3-epimerase / 生物工程学报

D-allulose-3-epimerase (DPEase) is the key enzyme for isomerization of D-fructose to D-allulose. In order to improve its thermal stability, short amphiphilic peptides (SAP) were fused to the N-terminal of DPEase. SDS-PAGE analysis showed that the heterologously expressed DPEase folded correctly in Bacillus subtilis, and the protein size was 33 kDa. After incubation at 40 °C for 48 h, the residual enzyme activity of SAP1-DSDPEase was 58%. To make the recombinant B. subtilis strain reusable, cells were immobilized with a composite carrier of sodium alginate (SA) and titanium dioxide (TiO2). The results showed that 2% SA, 2% CaCl2, 0.03% glutaraldehyde solution and a ratio of TiO2 to SA of 1:4 were optimal for immobilization. Under these conditions, up to 82% of the activity of immobilized cells could be retained. Compared with free cells, the optimal reaction temperature of immobilized cells remained unchanged at 80 °C but the thermal stability improved. After 10 consecutive cycles, the mechanical strength remained unchanged, while 58% of the enzyme activity could be retained, with a conversion rate of 28.8% achieved. This study demonstrated a simple approach for using SAPs to improve the thermal stability of recombinant enzymes. Moreover, addition of TiO2 into SA during immobilization was demonstrated to increase the mechanical strength and reduce cell leakage.

Hexavalent chromium reduction by immobilized cells of Bacillus sphaericus AND 303

Bacillus sphaericus AND 303, a Cr(VI)-resistant and reducing bacterium reported from serpentine outcrops of Andaman was evaluated for Cr(VI) reduction using immobilized cells under batch culture. Screening of inert matrices for entrapment of whole cells indicated that polyvinyl alchohol-alginate was the most effective one reducing 87.5% of 20 µM Cr(VI) in 24 h. The rate of chromate reduction was dependent on initial Cr(VI) and biomass concentrations. The PVA cell beads were recycled three times without cell leakage and disintegration. The reduction efficiency was improved in the presence of glucose and glycerol as electron donors leading to complete reduction. However, the presence of additional metal ions was inhibitory to Cr(VI) reduction. It could be emphasized that PVA-alginate immobilized cells of B. sphaericus AND 303 could be used as a continuous bioprocess in treating Cr(VI) contaminated effluents.

Immobilization of Bacillus megaterium MTCC 2444 by Ca-alginate entrapment method for enhanced alkaline protease production

Optimization of culture conditions and immobilization parameters for alkaline protease production was carried out by employing Bacillus megaterium MTCC2444. The partially purified enzyme was tested for its stability in the presence of oxidants, surfactants and commercial detergents. The optimum temperature, pH, incubation time and inoculum size were 55 ºC, 11, 48 h, 1 %, respectively. Calcium alginate was used as the immobilization matrix and the effects of gel concentration, bead size, age of immobilized cells, solidification period and initial biomass concentration on alkaline protease production and cell leakage were investigated. The results indicated that the immobilization was most effective with 4 % gel concentration, bead size of 3 mm, 24 h aged immobilized cells for a solidification period of 12 h at 1.5 % initial biomass concentration. The enzyme showed good stability in the presence of oxidants, surfactants and commercial detergents.

Produccion de beta-glucosidasas por cultivos de bacterias termofilas indigenas del altiplano boliviano / beta-Glucoside production by thermophilic bacteria indigenous the bolivian altiplano culture

Las beta-glucosidasas son enzimas que poseen actividad hidrolitica y transferasa o transglucosidasa. Tienen diversas aplicaciones; en la biosintesis de oligosacaridos, produccion de etanol utilizando residuos agricolas y en la industria de vinos. La aplicacion industrial, sin embargo, requiere estabilidad a temperaturas elevadas, por lo que los microorganismos termofilos tienen gran interes. El proposito de esta investigacion es el de optimizar el medio de cultivo anaerobio de bacterias termofilas, para aumentar la produccion de beta-glucosidasas. Esta enzima es producida por tres aislados bacterianos: FT3, 2B y P5 los cuales fueron aislados de la region andina de Bolivia. El aislado bacteriano FT3 mostro una actividad beta-glucosidasa de 0,35 [UI/mL]. Se tomaron como variables dentro de la optimizacion del medio de cultivo las fuentes de nitrogeno y de carbono, y el pH. Asi tambien se probaron dos sistemas de cultivo: celulas libres y encapsuladas. Empleando extracto de levadura como fuente de nitrogeno se obtuvo una actividad de 0,52 [UI/mL]. En la optimizacion del pH del medio de cultivo se obtuvo una actividad de 0,81 [UI/mL] a pH 5. Como fuente de carbono se eligieron los hidrolizados de paja de trigo y paja de quinoa lleg¨¢ndose a obtener actividades de 1,27 y 1,34 [UI/mL] respectivamente. Se establecio que la localizacion celular de la enzima beta-glucosidasa es extracelular y presenta estabilidad hasta una temperatura de 80 ºC y un pH de 7.

The beta-glucosidases possess hydrolytic and transferase activity or transglucosidase. They have various applications; such as biosynthesis of oligosaccharides, production of ethanol using agricultural residues and wine industry. However for industrial application, stability to high temperatures is needed. Therefore a great interesting in the thermophile microorganism study exist. The purpose of this research is to optimize the culture medium of thermophilic anaerobic bacteria to increase the production of beta-glucosidase. This enzyme is produced by three isolate bacterial FT3, 2B and P5 which were isolated from the Andean region of Bolivia. FT3 isolate showed beta-glucosidase activity of 0.35 [IU/mL]. In regards to the optimization of culture medium variables such as nitrogen source, carbon source and pH were taken into account and also the combination with free and encapsulated bacterial cells. Yeast extract was the selected source of nitrogen obtaining an activity of 0.52 [IU/ mL]. The optimal pH was 5 obtaining an activity of 0.81 [IU/mL]. The selected carbon source was the hydrolyzed wheat straw and quinoa straw obtaining activities of 1.27 and 1.34 [IU/mL], respectively. The cellular localization of beta-glucosidase enzyme is extracellular and provides stability to temperature of 80 ºC and stability at pH 7.

Degradation of h-acid by free and immobilized cells of Alcaligenes latus

Alcaligenes latus, isolated from industrial effluent, was able to grow in mineral salts medium with 50 ppm (0.15 mM) of H-acid as a sole source of carbon. Immobilization of Alcaligenes latus in Ca-alginate and polyurethane foam resulted in cells embedded in the matrices. When free cells and immobilized cells were used for biodegradation studies at concentration ranging from 100 ppm (0.3 mM) to 500 ppm (1.15 mM) degradation rate was enhanced with immobilized cells. Cells immobilized in polyurethane foam showed 100 percent degradation up to 350 ppm (1.05 mM) and 57 percent degradation at 500 ppm (1.5 mM). Degradation rate of Ca-alginate immobilized cells was less as compared to that of polyurethane foam immobilized cells. With Ca-alginate immobilized cells 100 percent degradation was recorded up to 200 ppm (0.6 mM) of H-acid and only 33 percent degradation was recorded at 500 ppm (1.5 mM) of H-acid. Spectral analysis of the products after H-acid utilization showed that the spent medium did not contain any aromatic compounds indicating H-acid degradation by A. latus.

Expression of XYL1 of Candida tropicalis in Pichia pastoris and Xylitol Fermentation by Immobilizing Cells / 中国生物工程杂志

XYL1 gene,which encodes xylose reductase with dual coenzyme activity from Candida tropicalis,was transformed into Pichia pastoris X-33 by expression vector pGAPZB.The recombination strain was immobilized in Ca-alginate beads and fermentation characterization is studied using corn cob hydrolysates.Fermentation conditions were as follow:initial pH value 6.0,30℃,initial cell concentration of 20%,the Liquid volume of 28%,rotation speed 130r/min.The average xylitol yield was 37.5% on the optimum condition.This result is expected to provide a new alternative method for producing xylitol on a large scale by bioconversion.

Photoproduction of hydrogen by photosynthetic bacteria from sewage and waste water.

Numerous prokaryotes, belonging to physiologically and taxonomically different groups, are able to produce hydrogen. Some photosynthetic bacteria have the property of light-dependent production of hydrogen from organic substrates. We isolated several photosynthetic purple and green bacteria from enrichment cultures made from the water of a waste-water pond of a cool-drink refilling station. After testing them for their ability to use various organic compounds as carbon source, and sulphide, thiosulphate and organic compounds as electron donor, we selected the fastest-growing isolate, a Rhodopseudomonas, for a study of its ability to produce molecular hydrogen in presence of light. Immobilized cells of this isolate produced significant amounts of hydrogen from both sewage and waste water.

Study on Degrading Phenol by Immobilized Ralstonia metallidurans CH34 / 微生物学通报

The subject of this study is to improve the ability of Ralstonia metallidurans CH34, a phenol-degrading strain with anti-heavy metal feature by immobilization.The optimal conditions for immobilizing bacterial cells were obtained with cross act experiment. The phenol-degrading efficiency of immobilized Ralstonia metallidurans CH34 was investigated. Results show that phenol-degrading efficiency of immobilized cells is obviously superior to that of the free cells; The ability of its resistance to heavy metal have been greatly improved; When additional carbon sources were added in the simulated waste water, the immobilized R. metallidurans CH34 were affected less than the free cells.

The Application of Immobilized Cells in ATP Biosynthesis / 微生物学通报

The process of ATP biosynthesis with immobilized cells were reviewed from three respects: the active biological material of catalysis, the form of immobilized cells and the bioreactor of immobilized cells. The developing trend of ATP production was discussed and the suggestion to its research in future was also presented.

Study on Immobilization of Petroleum Biodesulfurization Catalyst Agrobacterium tumefaciens UP-3 / 微生物学通报

Immobilization of the bacterium Agrobacterium tumefaciens UP-3 was studied in this paper. The results showed that the immobilized cells with the mixture of polyvinyl alcohol (PVA) and sodium alginate (SA) as the immobilizing carrier had good biodesulfurization characteristics; The optimum operation immobilization conditions were 4℃, the total concentration of PVA and SA being 7%(wt), and the concentration of cells being 0.05 g/mL. When DBT addition was 2.7 mmol/L, the DBT degradation of immobilized cells was above 60% while that of resting cells is 13%. The optimum degradation time and temperature of immobilized cells were 5d and 28℃～32℃, respectively.

A STUDY ON DECOLORIZATION OF DYES BY CELLS IMMOBILIZED IN BI-CARRIERS / 微生物学通报

Spreaded and immobilized the bacterial cells entraped in PVA on pieces of cotton cloth as porous carrier. The conditions for preparing immobilized cells were as below: cell concentration, 20 mg wet weight/ml; PVA concentration, 5%; spreading amount, 0.3ml/cm~2; immobilization for 12 hours in saturated boron acid solution. Then activated the immobilized cells in buffer containing dyes. Thus, immobilized cells with high decolorizing activity were obtained.In columns packed with immobilized cells, the decolorization efficiencies of continuous influent and intermissional influent were compared with each other. In twenty days, the decolorization rates were both higher than 90%; then the decolorization rate of continuous influent decreased to about 60% after 60 days while it still reached 80% in case of intermissional influent. The efficiency of the later was distinct higher than that of the former.