Seung Hoon Song

Selective biosorption of mixed heavy metal ions using polysaccharides

Although much research has been conducted on the separation of single species of heavy metal, the selective adsorption of two or more heavy metals in mixture is relatively little known. In this study, polysaccharide beads were prepared to selectively remove the targeted heavy metal ion from mixture. Among the biomasses, polysaccharide was examined due to its low cost and easy accessibility. In a single metal ion system, chitosan, λ-carrageenan, and alginic acid showed high affinity to mercury, copper, and lead, respectively. In the ion mixture, the same trend shown in the single metal ion solution was observed. The optimum electrolyte concentraion was investigated to adsorb the metal ion selectively, and it was possible to remove the targeted metal ion selectively with chitosan, alginic acid and λ-carrageenan at 1 mmol concentration of electrolyte. In order to demonstrate the feasibility of selective biosorption, two packed-bed reactors in series containing chitosan and alginic acid beads in each were studied and selective adsorption to Hg2+ and Pb2+, respectively, was observed.

Permeabilization ofOchrobactrum anthropi SY509 cells with organic solvents for whole cell biocatalyst

Permeabilization is known to overcome cell membrane barriers of whole cell biocatalysts. The use of organic solvents is advantageous in terms of cost, simplicity, and efficiency. In this study,Ochrobactrum anthropi SY509 was permeabilized with various organic solvents. Treatment with organic solvents resulted in lower permeability barriers due to falling out lipids of the cell membrane. Therefore, permeabilized cells showed higher enzyme activity with no cell viability. Among various organic solvents, 0.5% (v/v) chloroform was selected as the most efficient permeabilizing reagent. Changes in the cell membrane structure were observed and the residual amounts of phospholipids of the cell membrane were measured to investigate the mechanism of the improved permeability.

Elicitation of camptothecin production in cell cultures ofCamptotheca acuminata

Campothecin production was increased with elicitors, methyl jasmonate, jasmonic acid, yeast extract elicitor, and ferulic acid in suspension cultures ofCamptotheca acuminata. jasmonic acid was found to be the most efficient elicitor. Camptothecin production increased 11 times by using the optimum dosing concentration of jasmonic acid which was 50 μM. The kinetics of camptothecin accumulation in response to the treatment with jasmonic acid showed that the camptothecin accumulation reached the maximum value at 4 days after jasmonic acid dosing and then a rapid decrease in camptothecin accumulation was observed.

Novel sol-gel immobilization of horseradish peroxidase employing a detergentless micro-emulsion system

A novel sol-gel immobilization method employing a detergentless micro-emulsion system that consisted ofn-hexane/iso-propanol/water was developed and used to immobilize a horseradish peroxidase (HRP). Micro-sized gel powder containing enzymes was generated in the ternary solution without drying and grinding steps or the addition of detergent, therefore, the method described in this study is a simple and straightforward process for the manufacture of gel powder. The gel powder made in this study was able to retain 84% of its initial enzyme activity, which is higher than gel powders produced through other immobilization methods. Furthermore, the HRP immobilized using this method, was able to maintain its activity at or above 95% of its initial activity for 48h, whereas the enzyme activities of free HRP and HRP that was immobilized using the other sol-gel method decreased dramatically. In addition, even when in the presence of excess hydrogen peroxide, the enzyme immobilized using the novel sol-gel method described here was more stable than enzymes immobilized using the other method.

Characterization of membrane-bound nitrate reductase from denitrifying bacteriaOchrobactrum anthropi SY509

In this study, we have purified and characterized the membrane bound nitrate reductase obtained from the denitrifying bacteria,Ochrobactrum anthropi SY509, which was isolated from soil samples.O. anthropi SY509 can grow in minimal medium using nitrate as a nitrogen source. We achieved an overall purification rate of 15-fold from the protein extracted from the membrane fraction, with a recovery of approximately 12% of activity. The enzyme exhibited its highest level of activity at pH 5.5, and the activity was increased up to 70°C. Periplasmic and cytochromic proteins, including nitrite and nitrous oxide reductase, were excluded during centrifugation and were verified using enzyme essay. Reduced methyl viologen was determined to be the most efficient electron donor among a variety of anionic and cationic dyestuffs, which could be also used as an electron donor with dimethyl dithionite. The effects of purification and storage conditions on the stability of enzyme were also investigated. The activity of the membrane-bound nitrate reductase was stably maintained for over 2 weeks in solution. To maintain the stability of enzyme, the cell was disrupted using sonication at low temperatures, and enzyme was extracted by hot water without any surfactant. The purified enzyme was stored in solution with no salt to prevent any significant losses in activity levels.

Effect of aeration on denitrification byOchrobactrum anthropi SY509

Aeration was found to affect the biological denitrification byOchrobactrum anthropi SY509. Although cell growth was vigorous under 1 vvm of aeration and an agitation speed of 400 rpm in a 3-L jar fermentor, almost no nitrate was removed. Yet under low agitation speeds (100, 200, and 300 rpm), denitrification occurred when the dissolved oxygen was exhausted shortly after the inoculation of the microorganism.Ochrobactrum anthropi SY509 was found to express highly active denitrifying enzymes under anaerobic conditions. The microorganism also synthesized denitrifying enzymes under aerobic conditions (1 vvm and 400 rpm), yet their activity was only 60% of the maximum level under anaerobic conditions and the nitrate removal efficiency was merely 15%. However, although the activities of the denitrifying enzymes were inhibited in the presence of oxygen, they were fully recovered when the conditions were switched to anaerobic conditions.

Effects of electron donors on nitrate removal by nitrate and nitrite reductases

Effects of artificial electron donors to deliver reducing power on enzymic denitrification were investigated using nitrate reductase and nitrite reductase obtained fromOchrobactrum antropi. The activity of nitrite reductase in the soluble portion was almost the same as that in the precipitated portion of the cell extract. Nitrate removal efficiency was higher with benzyl viologen than with methyl viologen or NADH as an artificial electron donor. The turn-over numbers of nitrate and nitrite reductase were 14.1 and 1.9 μmol of nitrogen reduced/min·mg cell extracts, respectively when benzyl viologen was used as an electron donor.

Selection of mediators for bioelectrochemical nitrate reduction

The bioelectrochemical reduction of nitrate in the presence of various mediators including methyl viologen and azure A was studied using a 3-electrode voltammetric system. The catalytic potential for the reduction of the mediators was observed in the reactor, which for methyl viologen and azure A were −0.74 V and −0.32 V, respectively, with respect to the potential of Ag/AgCl reference electrode. This potential was then applied to a working electrode to reduce each mediator for enzymatic nitrate reduction. Nitrite, the product of the reaction, was measured to observe the enzymatic nitrate reduction in the reaction media. Methyl viologen was observed as the most efficient mediator among those tested, while azure A showed the highest electron efficiency at the intrinsic reduction potential when the mediated enzyme reactions were carried out with the freely solubilized mediator. The electron transfer of azure A with respect to time was due to the adhesion of azure A to the hydrophilic surface during the reduction. In addition, the use of the adsorbed mediator on conductive activated carbon was proposed to inhibit the change in the electron transfer rate during the reaction by maintaining a constant mediator concentration and active surface area of the electrode. Azure A showed better than nitrite formation than methyl viologen when used with activated carbon.

On-line monitoring of the denitrification process by measurement of NADH fluorescence

On-line monitoring method of the denitrification process was developed by NADH fluorescence measurements using the facultative microorganism, Ochrobactrum anthropi SY509. The NADH fluorescence signals showed a rapid drop and a rise at the initiation and termination points of the denitrification, respectively. This NADH fluorescence method could be applied successfully to the monitoring of the denitrification process in sequential batch and continuous operations while these rapid changes of fluorescence were not observed in a batch operation due to accumulation of some metabolites secreted from the microorganism.