The JS18 strain was isolated from an old tree forest and produced extracellular enzymes that decolorize synthetic melanin. Phylogenetic analysis, based on the internal transcribed spacer (ITS) sequence, indicate that JS18 belongs to the Trametes velutina species. JS18 demonstrated laccase activity but no manganese peroxidase or lignin peroxidase activity. Batch culture indicated that the melanin decolorization activity of JS18 strain originated from the laccase. Syringic acid and CuSO4 induced maximum laccase production, yielding 98 U/ml laccase activity after cultivation for 7 days at 25℃. T. velutina secretes an extracellular laccase in GYP medium, and this enzyme was purified using (NH4)2SO4 precipitation, Hi-trap Q Sepharose columns and gel filtration. The molecular weight of the purified enzyme was estimated to be 67 kDa using sodium dodecyl sulfate polyacrylamide gel electrophoresis. This enzyme produced 80% of its melanin decolorization activity within the first 24 h of evaluation in the presence of 1-hydroxybenzotriazole (HBT), while only about 4% of the melanin was decolorized in the absence of the mediator. The greatest decolorization was observed at 1.5 mM/l HBT, which decolorized 81% of the melanin within the first 24 h. The optimum pH and temperature for this decolorization were found to be 5.0 and 37℃, respectively. Our results suggest the possibility of applying HBT induced T. velutina JS18 laccase-catalyzed melanin decolorization.
The objective of this study was to optimize industrial-grade media for improving the biomass production of Weissella cibaria JW15 (JW15) using a statistical approach. Eleven variables comprising three carbon sources (glucose, fructose, and sucrose), three nitrogen sources (protease peptone, yeast extract, and soy peptone), and five mineral sources (K2HPO4, potassium citrate, ⳑ-cysteine phosphate, MgSO4, and MnSO4) were screened by using the Plackett-Burman design. Consequently, glucose, sucrose, and soy peptone were used as significant variables in response surface methodology (RSM). The composition of the optimal medium (OM) was 22.35 g/l glucose, 15.57 g/l sucrose, and 10.05 g/l soy peptone, 2.0 g/l K2HPO4, 5.0 g/l sodium acetate, 0.1 g/l MgSO4·7H2O, 0.05 g/l MnSO4·H2O, and 1.0 g/l Tween 80. The OM significantly improved the biomass production of JW15 over an established commercial medium (MRS). After fermenting OM, the dry cell weight of JW15 was 4.89 g/l, which was comparable to the predicted value (4.77 g/l), and 1.67 times higher than that of the MRS medium (3.02 g/l). Correspondingly, JW15 showed a rapid and increased production of lactic and acetic acid in the OM. To perform a scale-up validation, batch fermentation was executed in a 5-l bioreactor at 37℃ with or without a pH control at 6.0 ± 0.1. The biomass production of JW15 significantly improved (1.98 times higher) under the pH control, and the cost of OM was reduced by two-thirds compared to that in the MRS medium. In conclusion, OM may be utilized for mass producing JW15 for industrial use.
Kim, Hyun-Sang;Lee, Seong-Shin;Wi, Ji-Soo;Lee, Yoo-Kyoung
Korean Journal of Organic Agriculture
/
v.32
no.3
/
pp.289-298
/
2024
The objective of this study was to the effect of four medicinal plants (Rheum palmatum, Pharbitidis semen, Reynoutria japonica, Tribulus semen) supplementation on methane reduction and ruminal fermentation in in vitro batch culture method. Each medicinal plant was supplemented 5% on a substrate basis in the bottle, then filled with buffered rumen fluid. Incubation was conducted for 24 hours in a shaking incubator (39℃, 120 rpm). The ruminal pH values were not significantly different between the control and treatment groups. However, the digestibility of the feed was significantly higher in the group supplemented with medicinal plants than control group. Methane production (mL/g of digested dry matter) and total gas production (mL) was significantly lower in the treatment group compared to the control group in Tribulus semen group. Total volatile fatty acids concentration were significantly higher in all treatment groups than control group, and acetate concentration was significantly higher in all treatment groups than control group except for Rheum palmatum group. Propionate concentration was significantly higher in all treatment groups than control group, while butyrate concentration was significantly higher in Rheum palmatum group than control group. Ammonia nitrogen concentration was significantly higher in all treatment groups than control group. In conclusion, the addition of medicinal plants did not negatively impact rumen fermentation, and the results indicate that Tribulus semen has potential as a feed additive for reducing methane emissions.
A microorganism capable of producing high level of poly-3-hydoxybutyrate (PHB) from xylose was isolated from soil. The isolated strain J-65 was identified as Bacillus megaterium based on the morphological, biochemical and molecular biological characteristics. The optimum temperature and pH for the growth of B. megaterium J-65 were $37^{\circ}C$ and 8.0, respectively. The optimum medium composition for the cell growth was 2% xylose, 0.25% $(NH_4)_2SO_4$, 0.3% $Na_2HPO_4{\cdot}12H_2O$, and 0.1% $KH_2PO_4$. The optimum condition for PHB accumulation was same to the optimum condition for cell growth. Copolymer of ${\beta}$-hydroxybutyric and ${\beta}$-hydroxyvaleric acid was produced when propionic acid was added to shake flasks containing 20 g/l of xylose. Fermenter culture was carried out to produce the high concentration of PHB. In batch culture, cell mass was 9.82 g/l and PHB content was 35% of dry cell weight. PHB produced by B. megaterium J-65 was identified as homopolymer of 3-hydoxybutyric acid by GC and NMR.
Hyaluronic acid (HA) is an important macromolecule in medical and pharmaceutical fields. HA is a natural and linear polymer composed of repeating disaccharide units of β-1, 3-N-acetyl glucosamine and β-1, 4-glucuronic acid. This work aimed to confirm the structural characteristics and anti-inflammatory activities of HA and its chemically sulfated-HA. HA was produced from a fed-batch fermentation process using Streptococcus dysgalactiae in a 5 l bioreactor. HA was isolated water-soluble form (HA-WS) and water-insoluble form (HA-WI) from culture medium, and was obtained chemically sulfated-derivative (S-HA) that resulted in a 90% yield from HA-WI. The structural features of the sulfated- HA (S-HA) were investigated by FT-IR and 1H-NMR spectroscopy. The FT-IR and NMR patterns revealed the similarity in both the FTIR spectrum as well as NMR spectrum of both reference standard and purified HA from S. dysgalactiae. The anti-inflammatory activities of HA and S-HA were examined on LPS-induced RAW 264.7 cells. S-HA was significantly inhibited production of pro-inflammatory mediators such as nitric oxide (NO) and PGE2 and the gene levels of iNOS and COX-2, which are responsible for the production of NO and PGE2, respectively. Furthermore, S-HA also suppressed the overproduction of pro-inflammatory cytokine TNF-α (<80 pg/ml) and IL-6 (<100 pg/ml) compared to that of HA-WI. The present study clearly demonstrates that HA-S exhibits anti-inflammatory activities in RAW 264.7 macrophage cells.
A strain GP32 which produces a highly viscous extracellular polysaccharide was conducted with soil samples and identified as Pseudomonas species. The culture flask conditions for the production of extracellular polysaccharide by Pseudomonas sp. GP32 were investigated. The most suitable carbon and nitrogen source for extracellular polysaccharide production were galactose and (NH4)2SO4. The optimum carbon/nitrogen ratio for the production of extracellular polysaccharide was around 50. The optimum pH and temperature for extracellular polysaccharide production was 7.5 and 32℃, respectively. In batch fermentation using a jar fermentor, the highest extracellular polysaccharide content (15.7 g/l) was obtained after 70 hr of cultivation. The extracellular polysaccharide produced by Pseudomonas sp. GP32 (designated Biopol32) was purified by ethanol precipitation, cetylpyridinium chloride (CPC) precipitation, and gel permeation chromatography. Biopol32, which has an estimated molecular weight of over 3×107 datons, is a novel polysaccharide derived from sugar components consisting of galactose, glucose, gulcouronic acid and galactouronic acid in an approximate molar ratio of 1.85 : 3.24 : 1.00 : 1.42. The solution of Biopol32 showed non-Newtonian characteristics. The viscosity of Biopol32 exhibited appeared to be higher at all concentration compared to that of zooglan from Zoogloea ramigera. An analysis of the flocculating efficiency of Biopol32 in industry wastewater (food, textile, and paper wastewater) revealed chemical oxygen demand (COD) reduction rates 58.4-67.3% and suspended solid (SS) removal rates 82.6-91.3%. Based on these results, Biopol32 is a possible candidate for industrial applications such as wastewater treatment.
Effects of Zn, P and Fe on Cd uptake and accumulations by tomato (Lycopersicum esculentum Mill) and also their interactions on the uptake of Zn, Fe, Mn, P and Cd were investigated using batch type solution culture technique. Experiment 1 was a factorial scheme with 3 levels of Zn (0, 0.5, 2.5 ppm) and 3 levels of Cd (0, 0.2, 1.0 ppm). At 1.0 ppm Cd, significant yield reduction of dry matter and visual toxicity symptoms (yellowing and necrosis) of Cd was observed for all zinc levels. At this Cd level, increasing Zn treatment from 0 to 2.5 ppm increased Cd concentration from 199 to 235 ppm in leaves and from 124 to 145 ppm in stems. Similarly, Cd treatment did not suppress Zn uptake in leaves, and rather significantly increased in stems. Fe concentrations in leaves and stems were significantly reduced due to Cd treatment while Mn were increased by both Zn and Cd treatment. The results of experiment 2 with 3 levels of P (0.5, 2.0, 4.0m Mol) and 3 levels of Cd (0, 1.0, 2.0 ppm) in a factorial scheme also showed a growth reduction and visual toxic symptons from 1.0 ppm Cd level. Increasing P treatment tend to increase Cd concentrations in leaves and stems although it was not statistically significant. Increasing P concentration due to Cd treatment could be the 'concentration' effect as a result of reduced growth, while there was significant decrease in Fe concentration due to Cd treatment in spite of possible 'concentration' effect. Mn concentration was increased at 1.0 ppm Cd level and then dropped at 2.0 ppm Cd level. Zu concentration in leaves and stems showed significant increase as Cd treatment increased as observed in experiment 1. Experiment 3 had 3 levels of Fe (0.5, 1.0, 2.0 ppm) and 3 levels of Cd (0, 0.8, 1.6 ppm) treatments in a factorial design. Significant growth reduction and visual toxic symptoms as observed in experiment 1 and 2 were also observed from 0.8 ppm Cd level. Increasing Fe treatment obviously alleviated toxic symptoms, improved growth and significantly increased dry matter yield. At 0.8 ppm Cd treatment level, increasing Fe treatment from 0.5 to 2.0 ppm significantly decreased Cd concentration from 141 to 92 ppm in leaves and from 101 to 46 ppm in stems. At 1.6 ppm Cd treatment level the decrease was from 224 to 167 ppm in leaves and from 124 to 109 ppm in stems. As in the case of experiment 1 and 2, Fe concentration in leaves and stems were reduced as Cd treatment increased to 1.6 ppm at 0.5 and 1. 0 Fe treatment levels, whereas at 2.0 ppm Fe level, Cd treatment increased Fe concentration in leaves and stems showing significant interactions of Fe and Cd on Fe uptake. Cd effect on Zn and Mn showed similar results to experiment 1 and 2 and Fe treatments reduced Zn and Mn concentrations in plant tissue. The results of 3 experiments show that P and Zn did not manifest suppressive effect on Cd uptake, Fe significantly demonstrated it. Fe also alleviated Cd toxicity symptoms significantly in terms of visual symptoms and dry matter yield. Visual toxicity symptoms were definitely related to Fe status in plant tissue as well as possible physiological effect of Cd itself, and the results suggest that Fe requirement for normal growth increase as Cd element is present in plant tissue. Zn accumulated more in stems than in leaves whereas Cd, Fe and Mn showed the opposite trend in all experiments.
This study investigated the influence of temperature and $CO_2$ increase on phytoplankton growth and community structure during cold water season (spring) in Lake Paldang, Korea. Four experimental treatments of temperature and $CO_2$ manipulation were prepared in the laboratory batch culture: (1) Control; ambient low temperature ($6{\pm}2^{\circ}C$) and low $CO_2$ (air level, $400mgL^{-1}$), (2) T1; low temperature and high $CO_2$ ($800mgL^{-1}$), (3) T2; high temperature ($20{\pm}2^{\circ}C$) and low $CO_2$, (4) T3; high temperature and high $CO_2$. Algal growth experiment was carried out for 10 days under the light intensity of $70{\mu}mol\;m^{-2}s^{-1}$ (L :D=24 : 0). The level of pH decreased in both T1 and T3, due to dissolution of added $CO_2$. The dominant phytoplankton species of ambient water, Cyclotella meneghiniana succeeded to Fragilaria capucina var. gracilis in high-temperature treatment groups (T2 and T3). Cyanobacteria were very rare at the beginning of the experiment, while Oscillatoria limnetica appeared in only high-temperature groups (T2 and T3) at $6{\sim}7^{th}$ day. $CO_2$ addition in ambient temperature (T1) induced the highest phytoplankton growth, and thereby producing the highest average cell density of $3.27{\pm}0.33\;10^4\;cells\;mL^{-1}$, followed by T2 ($2.65{\pm}0.26\;10^4\;cells\;mL^{-1}$), T3 ($2.09{\pm}0.16\;10^4\;cells\;mL^{-1}$), and Control ($1.86{\pm}0.13\;10^4\;cells\;mL^{-1}$) (F=7.167, p=0.000). In summary, temperature increase changed the phytoplankton community structure and $CO_2$ increase promoted the phytoplankton growth during the cold spring season in Lake Paldang, suggesting a potential effect of climate change on freshwater phytoplankton.
The effluents of chemical and petroleum industries often contain non-biodegradable aromatic compounds, with phenol being one of the major organic pollutants present among a wide variety of highly toxic organic chemicals. Phenol is toxic upon ingestion, contact, or inhalation, and it is lethal to fish even at concentrations as low as 0.005 ppm. Phenol biodegradation has been studied in detail using bacterial strains. However, these microorganisms suffer from substrate inhibition at high concentrations of phenol, whereby growth is inhibited. A phenol-degrading bacterium, P21, was isolated from oil-contaminated soil. The phenotypic characteristics and a phylogenetic analysis indicated the close relationship of strain P21 to Rhodococcus pyridinovorans. Phenol biodegradation by strain P21 was studied under shaking condition. The optimal conditions for phenol biodegradation by strain P21 were 0.09% $KNO_3$, 0.1% $K_2HPO_4$, 0.3% $NaH_2PO_4$, 0.015% $MgSO_4{\cdot}7H_2O$, 0.001% $FeSO_4{\cdot}7H_2O$, initial pH 9, and $20-30^{\circ}C$, respectively. When 1,000 ppm of phenol was added to the optimal medium, the strain P21 completely degraded it within two days. Rhodococcus pyridinovorans P21 could grow in up to 1,500 ppm of phenol as the sole carbon source in a batch culture, but it could not grow in a medium containing above 2,000 ppm. Moreover, strain P21 could utilize toxic compounds, such as toluene, xylene, and hexane, as a sole carbon source. However, no growth was detected on chloroform.
This paper described the extraction/purification of $\beta$-carotene from recombinant E.coli and evaluation of anti-wrinkle activity of purified $\beta$-carotene. No significant differences in extraction yields were observed when hexane or isobutyl acetate was used. However, extraction from wet-cell cake resulted in 2-fold higher amount of $\beta$-carotene than that from dry cells. Disruption of 5 g-wet cells by ultrasonic homogenizer, acetone dehydration, extraction with isobutyl acetate resulted in 36 mg of $\beta$-carotene corresponding to 61.2% of recovery. The formation and separation of $\beta$-carotene crystal improved the purity. 633 mg of $\beta$-carotene crystal with 93% purity was obtained from 223 g/L of wet-cell cake harvested from 2.5-L fed-batch culture broth. The cultures of normal human primary fibroblast were performed to investigate the effect of $\beta$-carotene on cytotoxicity as MTT assay and anti-wrinkle activity as collagen synthesis assays. $1.7{\mu}M$ of $\beta$-carotene was found to be optimal concentration at which 1.4-fold higher amount of collagen was synthesized than that in absence of $\beta$-carotene. This indicates that highly purified $\beta$-carotene can be obtained from recombinant E.coli by applying simple method with less toxic solvent and can be used in functional cosmetics as anti-wrinkle agent.
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