Isolation,identification, culture conditions,and laccase production of white rot fungus北大核心CSCD

The white rot fungi are members of Basidiomycota, which can degrade lignin and form white rot. They are high producers of extracellular laccases. In the present study, pure culture strain of high-temperature and high-laccase production types (numbered as BUA-01) was isolated from the fruiting bodies of a white rot fungus collected in the campus of Beijing University of Agriculture. The taxonomic characteristic was determined based on morphological and ITS sequence analysis. Furthermore, the optimal culture conditions for the mycelia were determined, including carbon source, nitrogen source, C/N ratio, growth factors, temperature, and pH. Extracellular laccase production was investigated in liquid fermentation with different concentrations of Cu (CuSO4) as inducer. Decolorizing activity of the fermentation broth was assayed using three azo dyes: Evans blue, methyl orange, and eriochrome black T. The results showed that the strain possessed the highest homology toward Trametes hirsuta, with the homology ratio of 100% and the genetic distance of 0, suggesting that the strain BUA-01 belonged to the genus Trametes. The culture condition investigated revealed that the optimal condition for mycelia growth included the following: carbon source, starch; nitrogen source, soybean powder and yeast extract; C/N ratio, 40/1 and 10/1; temperature, 37 °C; and pH, 6.0-7.0. The assayed growth factors had no significant effect on mycelial growth. It demonstrated high laccase activity in liquid fermentation. The highest extracellular laccase activity of 1 081.33 ± 6.3 U/mL was observed in the broth with a Cu adjunction concentration of 0.25 mmol/L after a 96-h culture period. It was about 26-fold higher than that of the control group. The isolated strain exhibited significant decolorizing activity toward the azo dyes Evans blue, methyl orange, and eriochrome black T, with the decolorization rate at 12 h of 93.31% ± 0.16%, 92.37% ± 0.42%, 79.25% ± 0.64%, respectively. This suggests that the strain possesses potential applications in laccase production and dye degradation. © 2018 Science Press. All rights reserved.     

Isolation,identification, and biological characteristics of a wild Chaga mushroom北大核心CSCD

In this study, a pure culture strain (numbered as F1501) was obtained using tissue separation and purification methods from the sclerotia of Chaga mushroom from Russia. Further researches included studies on classification, optimum growth conditions of mycelia, extracellular polysaccharides from fermentation broth and their antioxidant activity, and artificial acclimation. According to the identification of the internal transcribed spacer (ITS) region, it was confirmed that F1501 was a species of the genus Inonotus and family Hymenochaetaceae, which had 99% similarity with Inonotus obliquus. F1501 was further classified as I. obliquus based on the morphological characteristics of its mycelia and sclerotia. The optimal carbon source, nitrogen source, C/N ratio, growth factor, temperature, and pH value for the growth of the F1501 strain mycelia were maltose, beef extracts, 10/1, B2, 28 °C, and 8.0, respectively. Liquid fermentation of F1501 was performed using PD media and a 10% inoculation amount at 28 °C and 150 r/min for 7 d. The content of polysaccharides in the fermentation broth was 476.32 mg/L, with a total antioxidant activity of 0.19 mmol/L (Trolox) and hydroxyl free radical-scavenging activity of 72.7%. Artificial acclimation study revealed that the fruiting body-like structure was obtained using cottonseed hulls as the main substrate. In the present study, a new strain of I. obliquus and its biological characteristics were explored, which could provide a theoretical basis for the artificial acclimation and development of wild mushrooms. © 2018 Science Press. All rights reserved.     

Mycelial culture of wild Inonotus hispidus parasitizing the jujube tree and the cultivation conditions of its fruit body on rice medium北大核心CSCD

Inonotus hispidus is a kind of rare medicinal fungus, and its natural resources are very scarce. Currently, the artificial cultivation technology of I. hispidus is not completely developed, and this reflects on its extremely low biological conversion rate and long cultivation period. In order to improve the bioconversion rate and shorten the production cycle of I. hispidus, we first analyzed the mycelia culture conditions of the collected I. hispidus, and then we further explore the method of domesticated cultivation of its fruiting body in rice medium. During the process of mycelial culture, the suitable temperature, pH, carbon source, and nitrogen source for mycelial growth were selected using the mycelial growth rate as index. During the domesticated cultivation of the fruiting body, the suitable culture medium for its growth was selected using the bioconversion rate as index. Screening results of mycelial culture conditions showed that the optimal culture conditions for the growth of mycelium of the wild I. hispidus were: temperature of 25 °C, initial pH of 6.0, glucose as the carbon source, and yeast extract powder as the source of nitrogen. The results of the domesticated cultivation showed that the biotransformation rate of I. hispidus was higher when using rice as the main medium substrate. The optimal cultivation conditions were: a 0.2% yeast extract content in the nutrient solution, a 1:1.6 ratio of rice to nutrient solution, and a 4 mL inoculum of the liquid strain. Under these conditions, it took about 4 days for the mycelium to grow over the cultivation medium. The time required for the differentiation of the primordium to form fruit bodies was about 20 days, and the bioconversion rate reached 28.70% ± 5.05%. The results of this study indicate the feasibility of using rice as the main substrate for the cultivation of I. hispidus, and it also provide new insights for the finding of new cultivation substrates for other rare medicinal fungi. © 2018 Science Press. All rights reserved.     

Screening and identification of cellulose degrading bacterium Bacillus siamensis BREC-11 with tolerance to furfural北大核心CSCD

Furfural is a toxic metabolic inhibitor that is created during the conversion of lignocellulose to produce fuel, which can retard fermentation and increase production costs. thus, it is important for lignocellulosic conversion that the ability of the strain to resist furfural stress be improved. A cellulose-degrading bacterium BREC-11 with tolerance to furfural was isolated from the intestinal tract of Omphisa fuscidentalis hampson larvae via the addition of furfural in the medium. Based on analyses of morphological observations, physiological and biochemical characterizations, and 16S rDNA sequences, strain BREC-11 was shown to represent a member of the genus Bacillus and was named B. siamensis BREC-11. to study the tolerance concentration of strain BREC-11, a wide range of furfural formaldehyde concentrations were tested and strain BREC-11 was shown to grow in the mineral medium containing furfural up to 3.5 g/L. Cellulase activity of strain BREC-11 was determined at the tolerable concentration of 3.5 g/L furfural after incubation at 30 ℃ and 150 r/min for 2 days. Results indicated that filter paper enzyme, CMC-Na enzyme, and β-glucosidase activity was 0.1 U/mL, 0.21 U/mL, and 0.07 U/mL, respectively. BREC-11 is a cellulose-degrading bacterium with resistance to furfural, which has potential application in future bio-refinery processes. © 2018 Science Press. All rights reserved.     

Isolation,identification and desulfurization mechanism of a sulfur-oxidizing bacterium with salt tolerance北大核心CSCD

Micro-organism with efficient desulfurization performance is a key factor in the biological desulfurization technology. This study aimed to seek such a sulfur-oxidizing strain and understand its desulfurization mechanism. Wastewater in a sewage station of natural gas purification plant was used to screen the sulfide-oxidizing strain, and to identify it based on sequence similarity analysis of 16S rDNA and the morphological characteristics. Thiosulfate was used as substrate for investigating the sulfur oxidation performance and salinity tolerance; the OD600, content change of thiosulfate, sulfate, sulfur, pH and total alkalinity in the cultural system were also investigated. The strain DS-B was found to share the highest sequence similarity with Thioalkalivibrio thiocyanoxidans ARh2, therefore determined as Thioalkalivibrio. At the optimum temperature of 35 °C for growth and degradation, the removal efficiency of thiosulfate could reach 98.7% after 7 days. Strain DS-B had strong resistance to thiosulfate, and the optimal concentration of S2O32- was 2 × 104 mg/L. The analysis for sulfur oxides showed that it could oxidize thiosulfate by the pathway of S2O32-→SO42- / S2O32- → S → SO42-. Therefore the strain DS-B is a sulfur-oxidizing bacterium with great application prospect for its strong salt tolerance and conspicuous removal capability for thiosulfate.     

Isolation,identification, and the biodegradation characteristics of a benzoylurea insecticides-degrading strain北大核心CSCD

A chlorbenzuron, diflubenzuron, and hexaflumuron-degrading bacterium strain M6, was isolated from the activated sludge of an insecticide factory. The strain was identified as Achromobacter sp. according to an analysis on the 16S rRNA gene sequences, morphological, and physiological characteristics. Strain M6 could degrade more than 91% of 100 mg/L chlorbenzuron, diflubenzuron, and hexaflumuron within 48 hours, which could act as the sole carbon source. Strain M6 showed more chlorbenzuron degradation at a temperature range between 25 and 40 ℃ and a pH range between 6.0 and 8.0. The optimal temperature and the initial pH of medium for chlorbenzuron degradation by strain M6 were 30 ℃ and 7.0, respectively; the maximum chlorbenzuron tolerated concentration of strain M6 was as high as 400 mg/L. Strain M6 hydrolyzed 4-acetaminophenol into a purple-red product. Moreover, an approximately 1.4 kb DNA fragment, which could be expressed into an amidase to degrade amide pesticides, was amplified from the genomic DNA of strain M6. The results preliminarily proved that 3 benzoylurea insecticides could be degraded because of strain M6 hydrolyzing their amide bonds. This study obtained a highly efficient degrading strain and provided new resources and valuable information on benzoylurea insecticide degradation. © 2018 Science Press. All rights reserved.     

Screening and degradation characteristics of a tetracycline-degrading bacterial strain北大核心CSCD

Tetracycline is widely used in livestock and poultry breeding industry, which can cause serious problems to the environment. Antibiotic pollution has become an important environmental issue. This study aimed to isolate and identify a well-functioning tetracycline-degrading bacteria strain from activated sludge and to investigate its optimum degradation conditions. The strain was identified through morphological features, Gram staining, and the sequence analysis of 16S rRNA. Furthermore, the temperature, initial pH of the medium, inoculation amount, and type of metallic salt were analyzed to investigate the tetracycline degradation performance of the isolated strain. Based on the single factor test, the method of response surface analysis was adopted to optimize the degradation condition. The strain was named TTC-1 and identified as Klebsiella pneumoniae. The optimum condition for tetracycline degradation was determined as follows: temperature of 34.4 °C, pH of 7.22, and MnSO4 concentration of 0.32 g/L. Under this optimum condition, the predicted tetracycline degradation rate was 93.77%, whereas the observed value was 94.26%. The experimental results showed that the proposed model had high accuracy. TTC-1 showed a good performance in degrading tetracycline, which can provide reference for the bacteria during the biological treatment of tetracycline containing wastewater. © 2018 Science Press. All rights reserved.     

Dynamics of bacterial community of Suillus granulatus mushroom shiro in Pine (Pinus tabuliformis) forests北大核心CSCD

To evaluate bacterial community variation in the mushroom shiro of Suillus granulatus during fruiting, we collected soil samples from the mushroom shiro in the pine (Pinus tabuliformis) forest of mountainous area in Beijing from May to November and evaluated the bacterial community using polymerase chain reaction - denaturing gradient gel electrophoresis (PCR-DGGE). Total soil DNA was extracted using a commercial soil DNA isolation kit. PCR amplification and DGGE were performed using bacterial universal primers 338F and 518R. The specific bands were excised from the gel and sequenced. The results revealed that soil bacterial community maintained considerably high level and changed seasonally with the mushroom fruiting. In total, 53 bands of DGGE profiles were sequenced and divided into 5 phyla (Acidobacteria, Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria and 22 genera (Acidobacterium, Aminobacter, et al). Species from Proteobacteria and Acidobacteria were the dominant bacterial groups sharing considerably high relative abundance, while class a-Proteobacteria was the most abundant group. The variation of the relative abundance of γ-Proteobacteria species was consistent with the mushroom fruiting season. The relative abundance of Acidobacteria species obviously increased before mushroom flush (in July). The fruiting of S. granulatus and the relative abundance of γ-Proteobacteria were correlated with each other. The present study provided a basis for conservation and domestication of mushroom S. granulatus.     

Adsorption of benzene and toluene from waste gas using activated carbon activated by ZnCl2

A series of activated carbons with high surface area were prepared from walnut shell using chemical activation with ZnCl2. In this research the carbonization stage was carried out at 500℃. The performance of the synthesized carbons evaluated in adsorption of benzene and toluene from waste gas. The influence of impregnation ratio on the characteristics of synthesized activated carbons as well as their adsorption capacity was investigated. The ratio of activation agent to walnut shell was selected in the range of 0.5-2.0 wt/wt. The synthesized activated carbons were characterized using XRD, SEM, BET and FTIR techniques. The highest activated carbon production yield was obtained at impregnation ratio of 1.5 wt/wt. The XRD analysis illustrated that peaks intensity decreased with increasing impregnation ratio showing that amorphous property of samples was increased. The SEM analysis revealed successful pore development in synthesized activated carbons obtained at high impregnation ratios. The surface area of the activated carbons increased with increasing impregnation ratio and its maximum value reached 2643 m2.g 1 at impregnation ratio of 2/1. FTIR analysis indicated that the relative amount of different acidic surface groups on synthesized carbons was a function of impregnation ratio. Experimental results for benzene and toluene adsorption showed a high potential of employing synthesized impregnated activated carbon for treatment of waste gas. Generally, the amount of VOC adsorbed on the surface was affected by physicochemical properties of synthesized activated carbons.     

Removal of Cu（ll） ions from aqueous solution by activated carbon impregnated with humic acid

Humic acid （HA） was impregnated onto powdered activated carbon to improve its Cu（II） adsorption capability. The optimum pH value for Cu（II） removal was 6. The maximum adsorption capacity of HAimpregnated activated carbon was up to 5.98mg.g-1, which is five times the capacity of virgin activated carbon. The adsorption processes were rapid and accompanied by changes in pH. In using a linear method, it was determined that the equilibrium experimental data were better represented by the Langmuir isotherm than by the Freundlich isotherm. Surface charges and surface functional groups were studied through zeta potential and FTIR measurements to explain the mechanism behind the humicacid modification that enhanced the Cu（II） adsorption capacity of activated carbon.     

The oligopeptides production by the feather-degrading bacterium Bacillus methylotrophicus H0北大核心CSCD

Due to the high nutritive value of oligopeptides and the waste of feather resources, this study aimed at screening efficient strains of bacteria able to rapidly degrade feathers and produce large quantities of value-added oligopeptides. In order to assess the potential yield of oligopeptides, the promising strain H0 was selected from 16 feather-degrading microorganisms. To identify the strain, we analyzed the morphological and physiological characteristics of different strains, and carried out a gene sequence analysis of their 16S rRNAs. A single factor experiment was used to promote feather degradation and oligopeptide production, and the characteristics of the oligopeptides produced were also analyzed by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS). The strain was identified as Bacillus methylotrophicus. The optimal initial pH and temperature for oligopeptide production were 11 and 40 °C, respectively. After 72 h of fermentation under these optimal conditions, the feathers were almost completely degraded, with a 38.19% of oligopeptides (accounting for 67.53% of the total soluble peptides) and a 11.11% of free amino acids produced. LC-MS/MS analysis indicated that the oligopeptides were mainly short peptides containing 5-10 amino acids, with a molecular mass (Mr) of less than 1 300. Moreover, the peptides were abundant in branched-chain amino acids, that might be responsible for the antioxidant property of the feather hydrolysate. Our results demonstrate the great capability of B. methylotrophicus H0 in feather degradation and oligopeptide production. This research provides a high-quality microorganism resource, and the scientific basis for the development of feather-derived oligopeptide products. © 2018 Science Press. All rights reserved.     

苹果茎沟病毒对梨树幼苗生长及过氧化物酶活性的影响

Based on growth level and peroxidase activity, a comparative study and paried-samples t test were made between vires-free Zaosu pear seedlings and the seedlings inoculated with apple stem grooving virus(ASGV). The results showed : ( 1 )virus-free Zaosu pear seedlings grew better than inoculated ones. Of the seedlings the height, diameter, branches and autumn braches differed statistically significantly; (2)the peroxidase activity of inoculated seedlings was higher than that of virus-free ones, and the peak of peroxidase activity occurred on about d 7 and d 24 after inoculation. Fig 1, Tab 1, Ref 9     

Protoplast preparation and regeneration conditions of Bacillus subtilis ZGL14 producing alkaline pectinase北大核心CSCD

The protoplast fusion technique is one of the important microbial breeding methods. The main factors that affect protoplast preparation and regeneration were investigated using Bacillus subtilis ZGL14 as the starter strain. The optimal conditions for protoplast preparation and regeneration were determined in this study by optimizing the cell age, lysozyme concentration, enzymolysis time, enzymolysis temperature, pH values of the osmotic pressure stabilizer, regeneration medium types, and culture type. The results indicated that the optimal condition for protoplast preparation of B. subtilis ZGL14 was as follows: 9 h of cell age, 0.2 mg/mL of lysozyme concentration, 10 min of enzymolysis time, 32 °C of enzymolysis temperature, and 7.0 of osmotic pressure stabilizer pH value. Under this condition, the protoplast formation and regeneration rates were > 98% and > 30%, respectively. A good protoplast regeneration effect can be achieved using the regeneration medium with sodium succinate as osmotic pressure stabilizer in combination with Ca2+. The single-layer culture was better than double-layer for regeneration. The optimal condition for protoplast preparation and regeneration of B. subtilis ZGL14 was determined in this study, which provides technical support for protoplast fusion breeding of B. subtilis. © 2018 Science Press. All rights reserved.     

Removal of 17β-estradiol in laccase catalyzed treatment processes

The removal of 17β-estradiol （E2） in laccase catalyzed oxidative coupling processes was systematically studied in this work. We focused on the influence of pH and natural organic matter （NOM） on the performance of the enzymatic treatment processes. It was found that the optimal pH for E2 removal was between 4 and 6. The removal of E2 was slightly inhibited in the presence of NOM. Enzymatic transformation of E2 was second-order in kinetics with first-order to both the concentrations of the enzyme and contaminant. Mass spectrum （MS） analysis suggested that coupling products were formed through radical-radical coupling mechanism. The results of this study demonstrated that laccase catalyzed oxidative coupling process could potentially serve as a treatment strategy to control steroid estrogens.     

Inhibitory kinetics of β-N-acetyl-D-glucosaminidase from Nile tilapia (Oreochromis niloticus) by cadmium北大核心CSCD

Cadmium (Cd2+) pollution in an aquatic environment can negatively affect certain reproductive parameters of aquatic animals. β-N-acetyl-D-glucosaminidase (NAGase) is considered to play an important role in the fertilization process. The aim of the present study was to investigate the effect of Cd2+ on the activity of NAGase purified f rom the testis of Nile tilapia, toward contributing new knowledge on the breadth of negative effects of Cd2+ for Nile tilapia production. The kinetic method of substrate reaction was used for this assessment, and an inhibitory model was established to study the kinetics of NAGase under inhibition by Cd2+. The results showed that Cd2+ could reversibly inhibit the enzymatic activity of NAGase, and the half-maximal inhibitory concentration was estimated to be 40.95 mmol/L. Cd2+ was found to be a competitive inhibitor of NAGase, and the inhibitory constant was determined to be 17.13 mmol/L. The microscopic rate constants of inactivation were also determined. Together, these findings demonstrate that Cd2+ is a reversible inhibitor that can competitively inhibit NAGase. These results may provide a theoretical foundation for further studies on the reproduction of tilapia. © 2018 Science Press. All rights reserved.     

Optimization of methyl orange removal from aqueous solution by response surface methodology using spent tea leaves as adsorbent

The effective disposal of redundant tea waste is crucial to environmental protection and comprehensive utilization of trash resources. In this work, the removal of methyl orange （MO） from aqueous solution using spent tea leaves as the sorbent was investigated in a batch experiment. First, the effects of various parameters such as temperature, adsorption time, dose of spent tea leaves, and initial concentration of MO were investigated. Then, the response surface methodology （RSM）, based on Box- Behnken design, was employed to obtain the optimum adsorption conditions. The optimal conditions could be obtained at an initial concentration of MO of 9.75 mg·L-1, temperature of 35.3℃, contact time of 63.8 min, and an adsorbent dosage 3.90 g· L-1. Under the optimized condi- tions, the maximal removal of MO was 58.2%. The results indicate that spent tea leaves could be used as an effective and economical adsorbent in the removal of MO from aqueous solution.     

Enhanced dewatering characteristics of waste activated sludge with Fenton pretreatment: effectiveness and statistical optimization

In this work, the enhanced dewaterabing characteristics of waste activated sludge using Fenton pretreatment was investigated in terms of effectiveness and statistical optimization. Response surface method （RSM） and central composite design （CCD） were applied to evaluate and optimize the effectiveness of important operational parameters, i.e., H202 concentrations, Fe2＋ concentrations and initial pH values. A significant quadratic polynomial model was obtained （R2= 0.9189） with capillary suction time （CST） reduction efficiency as the response. Numerical optimization based on desirability function was carried out. The optimum values for H202, Fe2, and initial pH were found to be 178 mg-g-1 VSS （volatile suspended solids）, 211mg.gI VSS and 3.8, respectively, at which CST reduction efficiency of 98.25% could be achieved. This complied well with those predicted by the established polynomial model. The results indicate that Fenton pretreatment is an effective technique for advanced waste activated sludge dewatering. The enhancement of sludge dewaterability by Fenton＇s reagent lies in the migration of sludge bound water due to the disintegration of sludge flocs and microbial cells lysis.     

Identification and assessment of environmental burdens of Chinese copper production from a life cycle perspective

The environmental burdens of Chinese copper production have been identified and quantified in the context of typical technologies, materials supplies and environmental emissions by a life cycle approach. Primary and secondary copper production using copper ores and scraps, respectively, were analyzed in detail. The flash and bath smelting approaches and the recycling of copper scraps were selected as representative copper production processes. A quantitative analysis was also conducted to assess the influence of material transport distance in copper production. Life cycle assessment （LCA） results showed that resources depletion and human health contribute significantly to environmental burdens in Chinese copper production. In addition, the secondary copper production has dramatically lower environmental burdens than the primary production. There is no obvious distinction in overall environmental burdens in primary copper production by flash or bath smelting approach. However, resources depletion is lower and the damage to human health is higher for flash smelting approach. Ecosystem quality damage is slight for both approaches. Environ- mental burdens from the mining stage contribute most in all life cycle stages in primary copper production. In secondary copper production, the electrolytic refining stage dominates. Based on the life cycle assessment results, some suggestions for improving environmental performance were proposed to meet the sustainable development of Chinese copper industry.     

Enhancement of MK-7 synthesis by engineered strains of Bacillus subtilis natto that overexpress men genes and by increasing available dissolved oxygen北大核心CSCD

In recent years, researchers have discovered novel physiological functions of vitamin K2. In addition to promoting blood clotting, it can prevent osteoporosis and cardiovascular disease and is expected to treat some tumors and Parkinson’s disease. Bacillus subtilis natto is a probiotic that has been approved by the U.S. Food and Drug Administration for use as a bioproducer of vitamin K2. Its product’s main form is MK-7, which has a long half-life in the human body and high bioavailability. Bacillus subtilis natto displays great potential for large-scale biological preparation of vitamin K2. In this study, the Sipizizen method of Bacillus subtilis transformation was optimized to make it suitable for molecular transformation of Bacillus subtilis natto. Vectors for overexpression of all 8 genes involved in the menadione synthetic pathway were constructed, and changes in MK-7 fermentation yield after transformation of Bacillus subtilis natto were investigated. Three enzymes were found to exert significant effects on MK-7 synthesis, namely isopentenyltransferase (MenA), 1,4-dihydroxy-2-naphthoyl-CoA synthase (MenB), and nornaphthoquinone methyltransferase (MenG). A modified strain (BN-pABG) with higher MK-7 productivity was obtained by concerted overexpression of menA, menB, and menG. In a 5 L bioreactor, MK-7 synthesis was further enhanced by optimizing oxygen supply. The final yield of MK-7 from the modified strain was 62.21 mg/L, 1.26 times higher than that of the original strain. These results show that combined overexpression of menA, menB, and menG strongly promotes MK-7 synthesis by Bacillus subtilis natto, and optimizing the oxygen supply conditions also promotes more robust MK-7 synthesis. © 2022 Authors. All rights reserved.