Polar Sciences

A cation exchange resin was added at different stages in the growth of Sorangium cellulosum cultures to remove excess ammonium, which has a negative influence on the production of the anticancer agent, epothilone. When 8 g L −1 of cation exchange resin were added at the mid-death phase, the ammonium removal reached 83% and the maximum production of epothilone was 14.3 mg L −1 , which was 2.6 times greater than that of the control culture.

Sulfate re-addition pH adjustment a b s t r a c t Biological hydrogen production by the green alga, Chlamydomonas reinhardtii can be induced in conditions of sulfur deprivation. In this study, we investigated the repeated and enhanced hydrogen production afforded by the re-addition of sulfate with monitoring of pH and concentration of chlorophyll and sulfate. Without adjustment of the pH, the optimal concentration of re-added sulfate was 30 mM for the hydrogen production. By the re-addition of 30 mM of sulfate and the adjustment of the pH during 4 cycles of repeated production, we obtained the maximum amount of 789 ml H 2 l À1 culture, which is 3.4 times higher than that of one batch production without adjustment of pH, 236 ml H 2 l À1 culture.

A fed-batch culture process followed by subsequent photoautotrophic induction was established for the high density culture of astaxanthin-rich Haematococcus pluvialis using a CO2-fed flat type photobioreactor under unsynchronized illumination. Fed-batch culture was performed with an exponential feeding strategy of the growth-limiting nutrients, nitrate and phosphate, concurrently with the stepwise supplementation of light depending on the cell concentration. During the growth phase, a biomass of 1.47 g/L was obtained at a biomass productivity of 0.33 g/L/day. Photoautotrophic induction of the well-grown vegetative cells was performed consecutively by increasing the light intensity to 400 μmol photon/m2/s, while keeping the other conditions in the CO2-fed flat type photobioreactor fixed, yielding an astaxanthin production of 190 mg/L at an astaxanthin productivity of 14 mg/L/day. The proposed sequential photoautotrophic process has high potential as simple and productive process for the production of valuable Haematococcus astaxanthin.

Aptasensor, an aptamer-based sensor for non-labeled detection of human immunoglobulin E (IgE), was developed using surface plasmon resonance (SPR). Various oligo(ethylene glycol) mixtures of different molar ratios of EG 6 -COOH and EG 3 -OH were used for the construction of self-assembled monolayers (SAM). Direct assays and modified sandwich assays over a 1.0 nM anti-IgE, aptamer-immobilized, gold chip, consisting of a 1:3 molar ratio of EG 6 -COOH:EG 3 -OH SAM, were investigated for real-time IgE detection. The linear ranges for both were 1.0-1000 ng/mL and the lowest amounts of detectable analytes in the samples were 3.44 and 2.07 ng/mL for direct and modified sandwich assays, respectively. These results for IgE detection suggest that the aptamer offers the possibility of a simple and realistic biosensor platform based on a SAM-fabricated SPR system. Furthermore, as an optic-biosensor, an SPR aptasensor could be used for the diagnosis of allergies.

A homogenous detection of pathogen (Giardia lamblia cysts) based on the catalytic growth of gold nanoparticles (AuNPs) has been studied. In this study, centrifugal filters were employed as tools to concentrate and separate the pathogen cells, and moreover amplify the detection signal. The catalytic growth of gold nanoparticles was verified to be positively related to gold seeds concentration. On this basis, homogenous detection of the pathogenic bacteria in liquid phase was established by means of conjugating antibody to gold seeds.

Pretreatment technology is a prerequisite to facilitate the release of sugars from a lignocellulosic biomass prior to fermentation. Recently, some pretreatment methods have been tried with ionic liquids, but they were still expensive and unpractical. In this study, an efficient pretreatment method using ammonia and ionic liquid was developed for the recovery of bio-digestible cellulose from a lignocellulosic byproduct, rice straw, and the increase of ionic liquid utilization. The combined use of ammonia and ionic liquid ([Emim]Ac) treatment exhibited a synergy effect for rice straw with 82% of the cellulose recovery and 97% of the enzymatic glucose conversion. This cooperative effect showed over 90% of the glucose conversion even with a reduced enzyme usage and incubation time. The ionic liquid was successfully recycled more than 20 times. The 20th-recycled ([Emim]Ac) showed 74% of the cellulose recovery and 78% of the glucose conversion to rice straw. Compared with the conventional pretreatment, our combined method for lignocellulosic biomass pretreatment was an economical and eco-friendly.

Gas sparging was found to be a useful technique to reduce hydrogen partial pressure in the liquid phase to enhance the hydrogen yields of strictly anaerobically fermentative bacteria. The effect of nitrogen (N 2 ) sparging on hydrogen yield was investigated in sterile and non-sterile conditions using a pure strain of the hyperthermophilic eubacteria, Thermotoga neapolitana with glucose or xylose as a carbon source. The maximum hydrogen accumulations reached 41% of the gaseous mixtures after 30-40 h. Two applications of N 2 sparging after the H 2 content in the headspace reached the maximum levels gave an increase of H 2 production by 78% from 1.82 to 3.24 mol H 2 /mol glucose and by 56% from 1.41 to 2.20 mol H 2 /mol xylose. This result suggested that the removal of the produced H 2 from the gas headspace of the limited-volume, closed culture vial when it achieves the maximum level of H 2 tolerance of the bacterium is a necessary technique to improve its H 2 yield.

In the present study, cultivation conditions and medium components were optimized using statistical design and analysis to enhance the production of Chi21702, a cold-active extracellular chitinase from the Antarctic bacterium Sanguibacter antarcticus KOPRI 21702. Identification of significant carbon sources and other key elements was performed using a statistical design technique. Chitin and glycerol were selected as main carbon sources, and the ratio of complex nitrogen sources to carbon sources was determined to be 0.5. Among 15 mineral components included in basal medium, NaCl, Fe(C6H5O7), and MgCl2 were found to have the most influence on Chi21702 production. The optimal parameters of temperature, initial pH, and dissolved oxygen level were found to be 25°C, 6.5, and above 30% of air saturation, respectively. The maximum Chi21702 activity obtained under the optimized conditions was 90 U/L. Through statistical optimization methods, a 7.5-fold increase in Chi21702 production was achieved over unoptimized conditions. Chi21702 showed relatively high activity, even at low temperatures close to 0°C. The information obtained in the present study could be applied to the production of cold-active endochitinase on a large scale, suitable for a process at low temperature in industry.

Ramalin (␥-glutamyl-N -(2-hydroxyphenyl)hydrazide), a novel compound, was isolated from the methanol-water extract of the Antarctic lichen Ramalina terebrata by several chromatographic methods. The molecular structure of ramalin was determined by spectroscopic analysis. The experimental data showed that ramalin was five times more potent than commercial butylated hydroxyanisole (BHA) in scavenging 1-diphenyl-2-picryl-hydazil (DPPH) free radicals, 27 times more potent in scavenging 2,2 -azino-bis (3-ethylbenzthiazoline-6-sulfonic acid free radicals (ABTS • + ) than the vitamin E analogue, trolox, and 2.5 times more potent than BHT in reducing Fe 3+ to Fe 2+ ions. Similarly, ramalin was 1.2 times more potent than ascorbic acid in scavenging superoxide radicals and 1.25 times more potent than commercial kojic acid in inhibiting tyrosinase enzyme activity, which ultimately leads to whitening of skin cells. Ramalin showed no or very little cytotoxicity in human keratinocyte and fibroblast cells at its antioxidant concentration. Furthermore, ramalin was assessed to determine its antioxidant activity in vivo. One microgram per milliliter ramalin significantly reduced the released nitric oxide (NO) and 0.125 g/ml ramalin reduced the produced hydrogen peroxide (H 2 O 2 ) in LPS (lipopolysaccharide)stimulated murine macrophage Raw264.7 cells. Considering all the data together, ramalin can be a strong therapeutic candidate for controlling oxidative stress in cells.

An endochitinase was previously purified and the gene was cloned from the psychrophilic Antarctic bacterium, Sanguibacter antarcticus (KCTC 13143). In the present study, recombinant endochitinase, rChi21702, was expressed using a yeast expression system (Pichia pastoris) and codon optimization. The expressed rChi21702 was purified by Phenyl-Sepharose column chromatography. Optimal expression yielded 1-mg purified enzyme from 1-L bioreactor culture. When p-NP-(GlcNAc) 2 was used as a substrate, the specific activity of the enzyme was determined to be 20 U/mg. In vitro assays and thin-layer chromatography demonstrated that the recombinant enzyme has endochitinase activity that produces diacetyl-chitobiose as a dominant end product when chitooligomers, colloidal chitin, and the chromogenic p-NP-(GlcNAc) 2 are used as substrates. Optimal activity for rChi21702 was observed at 37°C and a pH of 7.6. Interestingly, rChi21702 exhibited 63% of optimal activity at 10°C and 44% activity at 0°C. Taken together, the results indicate that rChi21702 has psychrotolerant endochitinase activity even after recombinant expression in yeast cells.

The Escherichia coli nar promoter is maximally induced under anaerobic conditions in the presence of nitrate ion or under anaerobic only conditions, depending on the genotype of the E. coli nar promoter. Previously, we found that the E. coli nar promoter has some desirable characteristics as an inducible promoter in the E. coli host strains. In this study, the E. coli nar promoter with lacZ gene at the downstream was cloned onto a broad-host-range Gram-negative vector, pBBR122. It was then induced in some other Gram-negative host strains, such as Agrobacterium, Pseudomonas, and Rhizobium, to determine whether the E. coli nar promoter could be used as an inducible promoter in these strains. From shake-flask experiments it was found that the wild-type E. coli nar promoter cloned onto pBBR122, pNW61, was suppressed under aerobic conditions in an Agrobacterium host strain, was partially induced under microaerobic only conditions, and was maximally induced under microaerobic conditions in the presence of nitrate ion. Whereas the mutant-type E. coli nar promoter cloned onto pBBR122, pNW618, was suppressed under aerobic conditions and was maximally induced under microaerobic conditions, regardless of the presence of nitrate ion. This kind of induction pattern observed for the E. coli nar promoters in the Agrobacterium host strain was similar to that observed for the E. coli nar promoters in the E. coli host strain. On the other hand, it was found that both of the E. coli nar promoters, pNW61 and pNW618, in a Pseudomonas host strain were partially induced under aerobic conditions and were maximally induced under microaerobic conditions, regardless of the presence of nitrate. Finally, it was found that both of the E. coli nar promoters in a Rhizobium host strain were minimally induced, regardless of the presence of oxygen or nitrate ion. Similar induction patterns for the three strains were also observed from fermentor experiments in which the dissolved oxygen (DO) level was tightly controlled. From an evolutionary point of view, the results from the three Gram-negative host strains indicate that the E. coli nar promoter system, including the promoter and regulatory proteins, was best conserved in the Agrobacterium host strain and the least conserved in the Rhizobium host strain. From an industrial point of view, the results indicate that the E. coli nar promoter system can be used as an oxygen-dependent inducible promoter in both Agrobacterium and Pseudomonas host strains.

The Escherichia coli nar promoter is maximally induced under anaerobic conditions in the presence of nitrate ion or under anaerobic only conditions, depending on the genotype of the E. coli nar promoter. Previously, we found that the E. coli nar promoter has some desirable characteristics as an inducible promoter in the E. coli host strains. In this study, the E. coli nar promoter with lacZ gene at the downstream was cloned onto a broad-host-range Gram-negative vector, pBBR122. It was then induced in some other Gram-negative host strains, such as Agrobacterium, Pseudomonas, and Rhizobium, to determine whether the E. coli nar promoter could be used as an inducible promoter in these strains. From shake-flask experiments it was found that the wild-type E. coli nar promoter cloned onto pBBR122, pNW61, was suppressed under aerobic conditions in an Agrobacterium host strain, was partially induced under microaerobic only conditions, and was maximally induced under microaerobic conditions in the presence of nitrate ion. Whereas the mutant-type E. coli nar promoter cloned onto pBBR122, pNW618, was suppressed under aerobic conditions and was maximally induced under microaerobic conditions, regardless of the presence of nitrate ion. This kind of induction pattern observed for the E. coli nar promoters in the Agrobacterium host strain was similar to that observed for the E. coli nar promoters in the E. coli host strain. On the other hand, it was found that both of the E. coli nar promoters, pNW61 and pNW618, in a Pseudomonas host strain were partially induced under aerobic conditions and were maximally induced under microaerobic conditions, regardless of the presence of nitrate. Finally, it was found that both of the E. coli nar promoters in a Rhizobium host strain were minimally induced, regardless of the presence of oxygen or nitrate ion. Similar induction patterns for the three strains were also observed from fermentor experiments in which the dissolved oxygen (DO) level was tightly controlled. From an evolutionary point of view, the results from the three Gram-negative host strains indicate that the E. coli nar promoter system, including the promoter and regulatory proteins, was best conserved in the Agrobacterium host strain and the least conserved in the Rhizobium host strain. From an industrial point of view, the results indicate that the E. coli nar promoter system can be used as an oxygen-dependent inducible promoter in both Agrobacterium and Pseudomonas host strains.

In an attempt to find a low-cost promoter system which could be used for producing recombinant proteins, a low oxygen inducible hmp promoter system in Bacillus subtilis (LAB1886) was characterized by studying the effects of cell density (OD600), glucose concentration, pH, dissolved oxygen (DO) level, nitrate and nitrite concentration on cell growth and β-galactosidase expression. The optimal cultivation strategy was found to be to grow the cells in the presence of nitrate to OD600 = 0.2 at a high DO level (80%), and then to induce the hmp promoter by lowering the DO level to 1–2%. As a result, the specific β-galactosidase activity increased continuously to the maximal value of 1750 Miller Units. Nitrite, as the apparent inducer of the hmp promoter, was produced from nitrate reduction, and had profound effects on cell growth and β-galactosidase expression. Copyright © 2006 Society of Chemical Industry

The Escherichia coli nar promoter is maximally induced under anaerobic conditions in the presence of nitrate ion or under anaerobic only conditions, depending on the genotype of the E. coli nar promoter. Previously, we found that the E. coli nar promoter has some desirable characteristics as an inducible promoter in the E. coli host strains. In this study, the E. coli nar promoter with lacZ gene at the downstream was cloned onto a broad-host-range Gram-negative vector, pBBR122. It was then induced in some other Gram-negative host strains, such as Agrobacterium, Pseudomonas, and Rhizobium, to determine whether the E. coli nar promoter could be used as an inducible promoter in these strains. From shake-flask experiments it was found that the wild-type E. coli nar promoter cloned onto pBBR122, pNW61, was suppressed under aerobic conditions in an Agrobacterium host strain, was partially induced under microaerobic only conditions, and was maximally induced under microaerobic conditions in the presence of nitrate ion. Whereas the mutant-type E. coli nar promoter cloned onto pBBR122, pNW618, was suppressed under aerobic conditions and was maximally induced under microaerobic conditions, regardless of the presence of nitrate ion. This kind of induction pattern observed for the E. coli nar promoters in the Agrobacterium host strain was similar to that observed for the E. coli nar promoters in the E. coli host strain. On the other hand, it was found that both of the E. coli nar promoters, pNW61 and pNW618, in a Pseudomonas host strain were partially induced under aerobic conditions and were maximally induced under microaerobic conditions, regardless of the presence of nitrate. Finally, it was found that both of the E. coli nar promoters in a Rhizobium host strain were minimally induced, regardless of the presence of oxygen or nitrate ion. Similar induction patterns for the three strains were also observed from fermentor experiments in which the dissolved oxygen (DO) level was tightly controlled. From an evolutionary point of view, the results from the three Gram-negative host strains indicate that the E. coli nar promoter system, including the promoter and regulatory proteins, was best conserved in the Agrobacterium host strain and the least conserved in the Rhizobium host strain. From an industrial point of view, the results indicate that the E. coli nar promoter system can be used as an oxygen-dependent inducible promoter in both Agrobacterium and Pseudomonas host strains.

The Escherichia coli nar promoter is maximally induced under anaerobic conditions in the presence of nitrate ion or under anaerobic only conditions, depending on the genotype of the E. coli nar promoter. Previously, we found that the E. coli nar promoter has some desirable characteristics as an inducible promoter in the E. coli host strains. In this study, the E. coli nar promoter with lacZ gene at the downstream was cloned onto a broad-host-range Gram-negative vector, pBBR122. It was then induced in some other Gram-negative host strains, such as Agrobacterium, Pseudomonas, and Rhizobium, to determine whether the E. coli nar promoter could be used as an inducible promoter in these strains. From shake-flask experiments it was found that the wild-type E. coli nar promoter cloned onto pBBR122, pNW61, was suppressed under aerobic conditions in an Agrobacterium host strain, was partially induced under microaerobic only conditions, and was maximally induced under microaerobic conditions in the presence of nitrate ion. Whereas the mutant-type E. coli nar promoter cloned onto pBBR122, pNW618, was suppressed under aerobic conditions and was maximally induced under microaerobic conditions, regardless of the presence of nitrate ion. This kind of induction pattern observed for the E. coli nar promoters in the Agrobacterium host strain was similar to that observed for the E. coli nar promoters in the E. coli host strain. On the other hand, it was found that both of the E. coli nar promoters, pNW61 and pNW618, in a Pseudomonas host strain were partially induced under aerobic conditions and were maximally induced under microaerobic conditions, regardless of the presence of nitrate. Finally, it was found that both of the E. coli nar promoters in a Rhizobium host strain were minimally induced, regardless of the presence of oxygen or nitrate ion. Similar induction patterns for the three strains were also observed from fermentor experiments in which the dissolved oxygen (DO) level was tightly controlled. From an evolutionary point of view, the results from the three Gram-negative host strains indicate that the E. coli nar promoter system, including the promoter and regulatory proteins, was best conserved in the Agrobacterium host strain and the least conserved in the Rhizobium host strain. From an industrial point of view, the results indicate that the E. coli nar promoter system can be used as an oxygen-dependent inducible promoter in both Agrobacterium and Pseudomonas host strains.

In an attempt to find a low-cost promoter system which could be used for producing recombinant proteins, a low oxygen inducible hmp promoter system in Bacillus subtilis (LAB1886) was characterized by studying the effects of cell density (OD600), glucose concentration, pH, dissolved oxygen (DO) level, nitrate and nitrite concentration on cell growth and β-galactosidase expression. The optimal cultivation strategy was found to be to grow the cells in the presence of nitrate to OD600 = 0.2 at a high DO level (80%), and then to induce the hmp promoter by lowering the DO level to 1–2%. As a result, the specific β-galactosidase activity increased continuously to the maximal value of 1750 Miller Units. Nitrite, as the apparent inducer of the hmp promoter, was produced from nitrate reduction, and had profound effects on cell growth and β-galactosidase expression. Copyright © 2006 Society of Chemical Industry