A Thermo-Active Laccase Isoenzyme From Trametes trogii and Its Potential for Dye Decolorization at High Temperature.

A thermo-activation and thermostable laccase isoenzyme (Lac 37 II) produced by Trametes trogii S0301 at 37°C was purified to apparent homogeneity by anionic exchange chromatography and sephadex G-75 chromatography, with 12.3% of yeiled and a specific activity of 343.1 U mg-1. The molecular weight of the purified Lac 37 II was estimated to be approximately 56 kDa in 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The optimal pH and temperature for the protein was 2.7 and 60°C, respectively. The purified Lac 37 II showed higher resistance to all tested metal ions and organic solvents except for Fe2+ and Cd2+ at 37°C and the activity of the purified Lac 37 was significantly enhanced by Cu2+ at 50 mM. The K cat , K m , and K cat /K m of Lac 37 II were 2.977 s-1, 16.1 µM, and 184.9 s-1 µM-1, respecively, in the condition of pH 2.7 and 60°C using ABTS as a substrate. Peptide-mass fingerprinting analysis showed that the Lac 37 II matched to the gene-deduced sequences of lcc3 in T. trogii BAFC 463, other than Lcc1, Lcc 2, and Lcc 4. Compared with laccase prepared at 28°C, the onset of thermo-activation of Lac 37 II activity occurred at 30°C with an increase of 10%, and reached its maximum at the temperatures range of 40-60°C with an increase of about 40% of their original activity. Furthermore, Lac 37 II showed the efficient decolorization ability toward triphenylmethane dyes at 60°C, with decolorization rates of 100 and 99.1% for 25 mg L-1 malachite and crystal violet in 5 h, respectively, when hydroxybenzotriazole (HBT) was used as a mediator. In conclusion, it is the first time to report a thermo-activation laccase from a thermophilic T. trogii strain, which has a better enzyme property and higher decolorization ability among fungal laccases, and it also has a further application prospective in the field of biotechnology.

Purification and characterization of alginate lyase from Sphingomonas sp. ZH0.

Alginate lyases degrade alginate in a beta-elimination reaction to produce oligosaccharides. Thus, alginate lyases are widely used in the food/pharmaceutical industries and are commercially valuable. In this study, four alginate lyase encoding genes were successfully cloned from Sphingomonas sp. ZH0. The expression systems of these alginate lyases were then constructed in Escherichia coli cells. The recombinant ZH0-I, ZH0-II, ZH0-III and ZH0-IV were purified from E. coli cells and were confirmed to be monomeric enzymes with molecular weights of approximately 91, 52, 67, and 113 kDa, respectively. The conditions for enzymes to have the highest specific lyase activities were 53.2 U/mg, 42 °C, pH 7.0 for ZH0-I, 103.9 U/mg, 47 °C, pH 6.5 for ZH0-II, 13.7 U/mg, 52 °C, pH 7.5 for ZH0-III, and 12.3 U/mg, 37 °C, pH 7.0 for ZH0-IV, respectively. These recombinant enzymes were stable over a pH range. Moreover, the enzymes were active in the absence of salt ions, and their activities were substantially reduced by the addition of HgCl2. ZH0-I, ZH0-II and ZH0-III belong to endotype alginate lyases, while ZH0-IV is an exotype alginate lyase. All types could degrade both poly-ß-d-mannuronate and poly-α-l-guluronate blocks, yielding alginate oligosaccharides as the main product. The Km and Vmax values were 0.51 mg/ml and 56.18 U/ml for ZH0-I, 0.47 mg/ml and 27.5 U/ml for ZH0-II, 0.55 mg/ml and 60.24 U/ml for ZH0-III, and 0.41 mg/ml and 5.53 U/ml for ZH0-IV, respectively. These features indicate that these alginate lyases are promising candidates for producing antioxidants from alginates in industrial applications.

A small proportion of litter-derived nitrogen is assimilated by plant biomass or immobilized in sediments regardless of harvest management as detected by 15N-labeled Phragmites litter in a constructed wetland.

Emergent aquatic macrophytes play an important role in the removal of nutrients in constructed wetlands (CWs). However, plant biomass supplies litter after the onset of senescence. Although litter-derived nitrogen (N) has been considered a nutrient source for the internal loading that may reduce CW performance, little is known about the quantitative N dynamics associated with litter decomposition. Thus, a controversial question remains about whether plant harvest is needed to manage CWs. In this study, we evaluated the decomposition and the fate of N derived from 15N-labeled Phragmites litter in a CW for 1 year. To simulate respective natural conditions, two treatments, including (1) a single winter harvest and (2) no harvest where the latter supplies a greater stem litterfall, were compared. Although the dry weight of the added stem litter was approximately 4.7 times larger in the no harvest plot than in the harvest plot, the total N content of the initial 15N-labeled litter was only 1.2 times higher in the no harvest plot than in the harvest plots because of the low N concentration in the stem litter. The litter functioned as a minor N sink within the first 6 months of decomposition, and it then shifted to functioning as a minor N source after 1 year of decomposition. The recovery of litter-derived N in the sediment and plant biomass was low (less than 10% of the initial litter N), and much of the remaining N might have been released into ambient water or lost through denitrification. Furthermore, our results suggested a potentially low contribution of litter-derived N to internal N loading for at least 1 year regardless of the harvest management treatment.

Effect of Phragmites japonicus harvest frequency and timing on dry matter yield and nutritive value.

Phragmites is a cosmopolitan perennial emergent macrophyte that is distributed worldwide. In recent years, Phragmites has attracted attention for its potential use as roughage. Given the increasing demand for feed and the number of constructed wetlands (CWs) vegetated with Phragmites, Phragmites is expected to play an important role in roughage production. Thus, it is vital to understand the effects of harvest timing and frequency on dry matter yield, nutritive value, and nitrogen (N) removal to establish appropriate vegetation management. In two CWs in Southwest China, four treatments with different harvesting frequencies were evaluated in monospecific areas of P. japonicus. The four treatments included no harvest, single harvest at 6 months, two harvests at 2 and 4 months, and three harvests at 2, 4, and 6 months. A sharp decline in the total digestible nutrients (TDN) concentration and the rate of increase in dry matter (DM) yield was associated with the heading timings, and the seasonal variations in TDN were likely influenced by carbohydrate accumulation in the stems. The three harvest treatment contributed to substantially improve the N and DM yields without decreasing the nutritive value but negatively affected the growth in the following year. Therefore, not only the combinations of harvest timing and frequency but also other management practices, including partial harvesting, may be needed to optimize CW performance and roughage production.

Estimation of potassium and magnesium flows in animal production in Dianchi Lake basin, China.

The objectives of this study were to estimate and evaluate potassium (K) and magnesium (Mg) budgets and flows of animal production in the basin of Dianchi Lake, China. Feed sampling and farmer interviews were conducted in field surveys. The supplies of K and Mg from local and external feeds and the retention, production and excretion of animals were calculated individually for dairy cows, fattening pigs, breeding sows, and broilers and laying hens. The K and Mg flows on a regional level were estimated using the individual budgets. At the individual level, in dairy cattle, the K and Mg supplied from local feeds accounted for large parts of the total nutrient intakes, whereas in the other animal categories most of the K and Mg in the feeds depended on external resources. Our findings also suggested that excessive Mg intake resulted in high Mg excretion and low use efficiency in dairy cattle and fattening pigs. At the regional level, the K and Mg amounts of manure produced and applied in the area (K: 339 and Mg: 143 t/year) exceeded those used as local feeds. Our results imply the animal production potentially increased the K and Mg loads in the regional agriculture system.

Timing of harvest of Phragmites australis (CAV.) Trin. ex Steudel affects subsequent canopy structure and nutritive value of roughage in subtropical highland.

In recent decades, constructed wetlands dominated by common reeds [Phragmites australis (CAV.) Trin. ex Steudel] have been utilized for treating nitrogen-rich wastewaters. Although plant harvest is a vegetation management in constructed wetlands for the purpose of improving nutrient removal, harvested biomass has become a problem in many places. The reed has attracted increasing interest for its potential as high-quality roughage for ruminants. Therefore, it is crucial to understand the effect of reed harvest timing on subsequent regrowth, reconstruction of canopy structure, and nutritive value of regrown biomass for roughage when defining an appropriate vegetation management in constructed wetlands. The shoots of common reeds were harvested in January (winter), March (spring), and May (early summer) in a free-water surface constructed wetland in southwest China. Harvesting in winter enhanced the shoot regrowth and concentrations of total digestible nutrients (TDN), probably due to vigorous translocations of nonstructural carbohydrates from rhizomes. Harvesting in spring and early summer decreased aboveground biomass, nitrogen (N) standing stock, and concentrations of TDN. From fifty to 110 days after harvest, the TDN had sharply declined to values similar to non-harvested stands. Thus, to obtain high-quality roughage, it is recommended that regrown shoots be harvested again within a year in the early growing stage after the first harvest in winter.

Estimation of nitrogen and phosphorus flows in livestock production in Dianchi Lake basin, China.

We assessed the nitrogen (N) and phosphorus (P) flows in intensified livestock production systems by investigating nutrient budgets and cycling in the basin of Dianchi Lake, one of the most eutrophic lakes in China. We conducted field surveys based on feed samplings and interviews of livestock farmers. The N and P in local and external feeds, animal body retentions, animal products and excretions were calculated at the individual level for dairy cattle, fattening pigs, breeding sows, broilers and laying hens. The N and P flows in the total livestock production system in the area were estimated by multiplying the individual N and P budgets by the number of animals. For the dairy and fattening pig productions, N and P supplied from local crops or by-products accounted for large parts of the inputs. For the other livestock categories, most of the N and P inputs depended on external resources. The N and P outputs through animal manure into the cropland were 287 and 66 kg/ha/year, respectively, which were higher than the N and P inputs into the livestock production systems from the cropland. The N and P loads from manure should be reduced for the establishment of sustainable agricultural production systems.

Bioethanol production from sodium hydroxide/hydrogen peroxide-pretreated water hyacinth via simultaneous saccharification and fermentation with a newly isolated thermotolerant Kluyveromyces marxianu strain.

In this study, bioethanol production from NaOH/H2O2-pretreated water hyacinth was investigated. Pretreatment of water hyacinth with 1.5% (v/v) H2O2 and 3% (w/v) NaOH at 25 °C increased the production of reducing sugars (223.53 mg/g dry) and decreased the cellulose crystallinity (12.18%), compared with 48.67 mg/g dry and 22.80% in the untreated sample, respectively. The newly isolated Kluyveromyces marxianu K213 showed greater ethanol production from glucose (0.43 g/g glucose) at 45 °C than did the control Saccharomyces cerevisiae angel yeast. The maximum ethanol concentration (7.34 g/L) achieved with K. marxianu K213 by simultaneous saccharification and fermentation (SSF) from pretreated water hyacinth at 42 °C was 1.78-fold greater than that produced by angel yeast S. cerevisiae at 30 °C. The present work demonstrates that bioethanol production achieved via SSF of NaOH/H2O2-pretreated water hyacinth with K. marxianu K213 is a promising strategy to utilize water hyacinth biomass.

Impact of plant harvest management on function and community structure of nitrifiers and denitrifiers in a constructed wetland.

Plant harvest is one of the most important management practices in constructed wetlands. In this study, we evaluated the impact of harvesting Phragmites australis (Cav.) Trin. ex Steudel on the activity and community structure of nitrifiers and denitrifiers in a free-water surface constructed wetland. The nitrifiers were targeted using bacterial and archaeal-amoA that encode ammonia monooxygenase, and the denitrifiers were targeted using nirK and nirS that encode the nitrite reductase. The community structures were evaluated using denaturing gradient gel electrophoresis. The potential nitrification and nitrate reduction rates were shown to be significantly higher in the harvested plant rhizosphere than in a non-harvested control plot. The potential nitrification rate positively correlated with the potential nitrate reduction rate and influenced the community structure of nirK. In addition, plant canopy developed differently after harvest and simultaneously changed the microclimate beneath the plant community. These results suggest that plant harvest management could change subsequent plant development and associated microenvironments, thereby affecting the function and community structure of nitrifiers and denitrifiers. Our study highlights the importance of plant harvest management within constructed wetlands to regulate the functions of nitrification and denitrification.

An ability of isolated strains to efficiently cooperate in ethanolic fermentation of agricultural plant refuse under initially aerobic thermophilic conditions: oxygen deletion process appended to consolidated bioprocessing (CBP).

For bioconversion of bean curd refuse, a processing by-product of bean curd, ethanol-producing anaerobic thermophiles (strains kpu03 and kpu04) were newly isolated. Both of them degraded hemicellulose, but not cellulose at all. Phylogenetically, strains kpu03 and kpu04 belong to the Clostridium and Thermoanaerobacterium genus, respectively. Aerobic thermophiles degrading cellulose were also isolated newly. Among them, strain kpuB3 particularly enhanced ethanol production by anaerobic strain kpu04 in the aerobic bean curd refuse medium. Strain kpuB3 belongs to the Geobacillus genus phylogenetically. The co-culture also significantly reduced CH(3)SH production, leading to the prevention of offensive odor. These results demonstrate that cellulolytic aerobe cooperated with hemicellulolytic anaerobe in ethanolic fermentation by not only synergistic effect but also deletion of oxygen from the vessels, providing a new model of oxygen deletion process appended to consolidated bioprocessing (CBP).

Detection of methanogens and proteobacteria from a single cell of rumen ciliate protozoa.

Rumen ciliate-associated bacteria and methanogenic archaea were analyzed by a 16S rRNA gene retrieved from a single cell of Polyplastron multivesiculatum, Isotricha intestinalis, and Ophryoscolex purkynjei. Rumen fluid was taken from a ruminally fistulated goat to prepare a ciliate fraction. Ciliate mixtures were incubated under mixtures of antibiotics for 48 h to eliminate extracellular bacteria. Individual cells of rumen ciliates were selected under microscopic observation after fixation with ethanol. Bacterial and archaeal 16S rRNA gene sequences were retrieved from each cell of three genera of ciliate. Two archaeal sequences related to Methanobrevibacter smithii were distributed to nearly all ciliate cells tested. These two methanogenic archaea were likely to be endosymbiotic methanogens commonly carried by the rumen ciliate, although some other sequences similar to the other genera were detected. A range of proteobacteria was retrieved from cells of P. multivesiculatum. Some sequences showed similarities to the previously known endosymbiotic proteobacteria. However, there were no proteobacteria that were carried by all the ciliate cells tested.