Characteristics of Biogenic Silica and Its Effect on Reservoir in Wufeng-Longmaxi Shales

四川盆地上扬子板块龙马溪组古生物与页岩气成藏的关系四川盆地作为我国西部重要的含油富气盆地,下古生界发育两套海相优质烃源岩(下寒武统牛蹄塘组页岩,下志留统龙马溪组页岩)。

本文重点研究四川盆地及其周缘龙马溪组页岩气成藏地质条件,预测其勘探前景。

研究区龙马溪组主要为深海陆棚、浅海深水盆地、斜坡相沉积的一套以黑色炭质页岩、放射虫硅质泥岩、粉砂质泥岩、泥质粉砂岩为主的细粒沉积物。

岩石中富含丰富的笔石,沉积物源来自于汉南古陆、川北隆起、川中古隆起,前两者是主要物源区。

龙马溪组烃源岩主要发育在龙马溪组下部,生烃条件优越,研究区存在镇巴―观音、云阳―石柱―观音桥、宜宾―泸州三个烃源岩厚度分布优势区,烃源岩厚度介于20～120m之间;有机质类型好,以I―II干酪根为主;有机质丰度高,TOC为1.0%～5.0%;有机质热演化程度高,存在川东、川东北、鄂西渝东、川南4个高热演化区,Ro值为1.2%～4.3%,其中川南的高热演化与259Ma年的热液活动有关;川东北的高热演化与沉积后期的深埋藏有关。

研究区多期构造运动,特别是燕山期―喜马拉雅期褶皱隆升导致龙马溪组高角度裂缝发育,有效增大泥页岩储集空间,同时有利于页岩中吸附态天然气的解吸。

龙马溪组中存在多种孔隙类型,包括有机质粒内微孔;伊利石片状微孔隙、微裂隙;方解石、白云石及石英等粒间微孔;斜长石粒间溶孔及云母片状孔隙、自生莓状黄铁矿晶间孔等;页岩中孔隙形成机理包括成岩过程中的矿物多相转变、不稳定矿物溶蚀、有机质生烃演化等。

龙马溪组烃源岩与Barnett页岩具有相似的沉积地质背景、有机质类型(I～II型干酪根)和生烃演化史;但龙马溪组埋藏较深(1600～4200m),相对富集黏土矿物、热演化程度更高(普遍Ro2%),较快的隆升速率(44m/Ma),较低的含气量(1.73～3.28m~3/t)。

龙马溪组作为川东石炭系古油藏(140×108t)和气藏群(2575.3×10~8m~3)烃源岩,曾经高效排烃。

Journal of Hazardous Materials 171 (2009) 1051–1057Contents lists available at ScienceDirectJournal of HazardousMaterialsj o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /j h a z m atThe characteristics of enriched nitrifier culture in the degradation of selected pharmaceutically active compoundsNgoc Han Tran a ,∗,Taro Urase b ,Osamu Kusakabe aa Department of Civil Engineering,Tokyo Institute of Technology,Ookayama 2-12-1,Meguro,Tokyo 152-8552,Japan bSchool of Bioscience and Biotech,Tokyo University of Technology,Katakura 1401-1,Hachioji,Tokyo 192-0982,Japana r t i c l e i n f o Article history:Received 21January 2009Received in revised form 22May 2009Accepted 19June 2009Available online 27 June 2009Keywords:Activated sludge DegradationEnriched nitrifier culture Pharmaceuticalsa b s t r a c tThe biodegradation of 10selected pharmaceuticals by enriched nitrifier cultures with ammonia oxidizing activity of 30mg NH 4-N/g MLVSS h was investigated under various initial operating conditions such as in the presence of different growth substrates and inhibitors.The enriched nitrifier culture showed higher degradation of the target pharmaceuticals than the conventional activated sludge.The degradation effi-ciency of persistent pharmaceuticals such as clofibric acid (CA),diclofenac (DCF),carbamazepine (CBZ),and propyphenazone (PPZ)was increased with the increase in the ammonium concentration.A higher removal efficiency of CA,DCF,CBZ and PPZ was obtained when organic substrates were added.The contri-bution of autotrophs and heterotrophs in the biotransformation of the pharmaceuticals by the enriched nitrifier culture was successfully estimated by the addition of inhibitors.Experimental results showed that the high degradation of IBP and partial degradation of other selected pharmaceuticals were observed in the presence of allylthiourea (ATU),an ammonia monooxygenase inhibitor,reflecting the activity of heterotrophic bacteria,while the results with and without ATU addition showed that the contribution of the nitrification in the degradation of most pharmaceuticals was also dominant.The results suggest that nitrification can enhance the biotransformation of pharmaceutical substances.© 2009 Elsevier B.V. All rights reserved.1.IntroductionThe occurrence and fate of pharmaceutically active compounds (PhACs)in the water environment have been recognized as one of the emerging issues in environmental chemistry due to their poten-tial to cause undesirable ecological and human health effects [1,2].PhACs have been detected ubiquitously in water environments and water distribution systems because of their high persistence and low adsorption properties.For example,carbamazepine has been detected in groundwater [3]and diclofenac was also found in tap water in Berlin [4].The removal of PhACs in water and wastewa-ter treatment is essential to prevent environmental contamination and possible adverse effects.Because these chemicals are often present in wastewater at concentrations in the low ␮g/l range or less [5,6]they cannot support cell growth and activity of organisms capable of mineralizing these compounds in biological wastewater treatment.The biodegradation of PhACs in wastewater is likely to be due to cometabolic activity.This means that PhACs can only be degraded∗Corresponding author.Tel.:+81357342798;fax:+81357343577.E-mail address:hantn04779@ (N.H.Tran).in the obligate presence of a primary substrate (growth support-ing substrate).Published reports on the cometabolism of PhACs are currently limited.Several studies on the removal of xenobi-otics have shown that the degradation can be enhanced by the augmentation through the addition of organic carbon sources or other nutrient substances such as nitrogen,and phosphate as well as mineral constituents [7,8].It was also suggested that primary substrates not only serve to sustain biomass production but also act as an electron donor for the cometabolism of the non-growth substrate [9].Several recent studies reported that the ammonia oxi-dizing bacteria (AOB)in nitrifying activated sludge system were responsible for the elimination of these chemicals in wastew-ater [10,11].The cometabolism of PhACs may be important in the degradation of PhACs because AMO (ammonium monooxy-genase)has a relatively wide spectrum for the degradation of substrates.Therefore,in this study in order to clarify the role of nitrifica-tion in the degradation of PhACs,an enriched nitrifier culture was prepared from activated sludge.The objectives of this study were to investigate the effect of supplement of the primary substrates (ammonium and organic substrates)on the biodegradability of the selected PhACs by enriched nitrifier culture and estimate the role of nitrification in the degradation of PhACs in biological wastewater treatment.0304-3894/$–see front matter © 2009 Elsevier B.V. All rights reserved.doi:10.1016/j.jhazmat.2009.06.1141052N.H.Tran et al./Journal of Hazardous Materials 171 (2009) 1051–10572.Materials and methods 2.1.Chemicals and mediaThe chemicals used in this study were purchased from Kanto-Kagaku,Japan.The composition of mineral-salts medium (MSM)was:Na 2CO 31514mg/l;MgSO 4·7H 2O 41.6mg/l;CaCl 2·2H 2O 50mg/l;NaH 2PO 450.5mg/l;K 2HPO 4·3H 2O 75.6mg/l;CuSO 4·5H 2O 0.5mg/l;FeCl 3·6H 2O 0.5mg/l and ZnSO 4·H 2O 0.5mg/l.The MSM was used with other supplements such as ammonium,organic substrates and target PhACs according to the experimental purposes.The target compounds used in this study were 10selected pharmaceutical compounds clofibric acid (CA),gemfibrozil (GFZ),ibuprofen (IBP),fenoprofen (FEP),ketoprofen (KEP),naproxen (NPX),diclofenac (DCF),indomethacin (IDM),propyphenazone (PPZ)and carbamazepine (CBZ).Table 1shows the chemicalstructures and physicochemical properties of these compounds [12].Mixed stock solution of 1g/l of the target pharmaceuticals was prepared in methanol and introduced in the MSM as necessary.2.2.Enrichment of nitrifying bacteriaActivated sludge with a low nitrifying activity of less than 5mg NH 4-N/g MLSS h was collected from a nitrification tank at a munic-ipal wastewater treatment plant in Japan.From this collection,nitrifiers were enriched in fill-and-draw operation with a 2d-cycle in a 2-l reactor at 30◦C for more than 2months.At the start of enrichment,activated sludge and the MSM solution supplemented with ammonium were used as enrichment medium.The ammo-nium concentration was gradually increased from 100to 300mg NH 4-N/l during the enrichment period depending on the growth in the nitrifying activity of the sludge.The pH in the reactor was con-Table 1The physicochemical properties and chemical structure of the target compounds by Ternes and Joss [12].Target compoundsChemical structureMol.weightlog K owp K aHenry’s coefficientCA 241.65 2.57 3.02.19×10−8GFZ 250.34 4.8n.a.n.a.IBP 206.29 3.97 4.911.50×10−7FEP 242.28 3.9 4.5n.a.KEP 254.29 3.12 4.52.12×10−11NPX 230.27 3.2 4.23.39×10−10DCF 296.16 4.5 4.154.73×10−12IDM 357.80 4.27 4.503.13×10−14PPZ 230.31 1.94–1.84×10−9CBZ 236.28 2.45–1.08×10−10n.a.:data not available.(–):not applicable.N.H.Tran et al./Journal of Hazardous Materials171 (2009) 1051–10571053trolled at7.5–8.0using NaHCO330g/pressed air was used to aerate the culture to maintain a dissolved oxygen level of6.0mg O2/l or higher.Conventional activated sludge(CAS)taken from another municipal wastewater treatment plant in Japan was also used in the experiment for a comparison with nitrifying activated sludge (NAS).2.3.Batch degradation experiments of PhACsSterilized300ml Erlenmeyerflasks were eachfilled with100ml of MSM supplemented with various initial ammonium or sodium acetate concentrations depending on the experimental purposes. The target pharmaceuticals in methanol solution were then added to theflasks to achieve an initial concentration of100␮g/l.The amount of the biomass in theflasks was adjusted to achieve an MLSS concentration of1000mg/l at the beginning of all batch tests.The pH of the culture medium was kept at approximately7.5–8.0using NaHCO330g/l during cultivation period.The suspension in the flasks was cultivated in a temperature-controlled incubator at30◦C. Shaking ensured a sufficient supply of oxygen to keep the dissolved oxygen concentration higher than3mg/l.To suppress the degrading activities of PhACs by ammonia oxidation,10mg/l of allylthiourea (ATU)was added as the AMO(ammonia monooxygenase)inhibitor [13]with the ammonium-supplemented MSM in the case of the inhibition experiments for nitrifiers.The experiments with the full inhibition of biological activities were also carried out to distinguish pure adsorption onto sludge from biodegradation.The inactivation of sludge was performed with the addition of sodium azide(NaN3) at0.2%weight to volume(w/v)into the ammonium-supplemented MSM.During the batch degradation experiments,the target com-pounds and NH4-N,NO2-N,NO3-N,and COD concentrations were measured.All batch experiments were implemented in triplicate.2.4.AnalysesThe concentrations of the target pharmaceutical substances such as CA,GFZ,IBP,FEP,KEP,NPX,DCF,and IDM were measured by the method using gas chromatography–mass spectrome-try(GC/MS)after solid phase extraction and pentafluorobenzyl derivatization suggested by Koutsouba et al.[14]with slight modi-fications.Solid phase extraction disks(C18,47mm disk typefilter, 3M)were used to concentrate100ml of water sample to which two surrogates,2,3-dichlorophenoxyacetic acid and chrysene-d12, and hydrochloric acid for the pH adjustment at2were added. After the extraction,2,4-dichlorobenzoic acid was added to check the recovery in the pentafluorobenzyl derivatization step.GC/MS (Shimadzu,QP–5000)with DB-5MS column was used for the quan-tification of the derivatized material in toluene solution.PPZ and CBZ,which require no derivatization in the analysis,were quanti-fied in the same chromatogram.Concentrations of NH4-N,NO2-N, NO3-N,and COD after heating with K2Cr2O7solution were deter-mined using the colorimetric tests following the protocol of HACH DR-2010.3.Results and discussion3.1.Enrichment of nitrifying bacteriaTo obtain a nitrifier-dominated population in the sludge,the enrichment was performed for more than2months.Ammonium oxidizing activity estimated from the disappearance rate of ammo-nium gradually increased and achieved30mg NH4-N/g MLVSS h after2months of enrichment.During the experimental period, the nitrifying activity of this sludge was maintained at around 25–30mg NH4-N/g MLVSS h.The volatile fraction of the nitrifying activated sludge was70±10%of total suspended solids.This nitrify-ing activated sludge(NAS)was used for the following experimental purposes.3.2.Degradation kinetics of selected pharmaceuticals in batch experimentsFig.1shows representative results obtained for the removal of selected pharmaceuticals in batch experiments.The batch experi-ment shown in Fig.1was performed with enriched nitrifier culture at an initial concentration of100␮g/l for all selected pharmaceu-ticals and NH4-N100mg/l.Exponential decreases in concentration over time were observed for all target compounds.In previous batch degradation studies using activated sludge spiked with selected pharmaceuticals it has been reported that the degradation of pharmaceuticals in the water phase can be described by a pseudo-first-order degradation kinetics[15–18]as given in Eq.(1):dC wdt=−k biol·X·C w(1) where C w is the concentration of the target substances in the water phase(␮g/l),t is the time(day),k biol is thefirst-order biodegrada-tion constant(l/g MLSS day),and X is the mixed liquor suspended solids concentration(g MLSS/l).Rearranging Eq.(1),the following equation is obtained,lnC wC o=−k biol X·t(2)where C o is the initial concentration of the selected pharmaceuti-cals in the water phase(␮g/l).This equation suggests that a plot of ln(C w/C o)versus X·t yields a straight line andfirst-order biodegra-dation constant k biol is obtained from the slope of the straight line by applying the least-squares method.Thefirst-order biodegrada-tion constants of selected pharmaceuticals in the water phase were calculated and are shown in Table2.The comparison of biodegradation constants shows that IBP was removed rapidly.This result is consistent with previous publica-tions[15–17,19].The estimated k biol for IDM,CBZ and DCF was slightly higher than those reported in the literature[16–18],while the calculated k biol for CA,GFZ,FEP,and IBP was significantly smaller than those reported by Joss et al.[16].This difference is probably due to aspects of the sludge composition such as biodiversity of biomass, types of primary substrates,fraction of active biomass withinthe Fig.1.Changes in normalized concentrations of selected pharmaceuticals during batch experiment using enriched nitrifier culture(NAS).1054N.H.Tran et al./Journal of Hazardous Materials 171 (2009) 1051–1057Table 2First-order biodegradation constants k biol of selected pharmaceuticals observed in batch experiments with enriched nitrifier culture (NAS).Target compoundBiodegradation constants (k biol )NAS (l/g MLSS day)aCAS (l/g MLSS day)b MBR (l/g SS day)c CAS (l/g SS day)d MBR (l/g SS day)e CA 0.03–0.070.0120.1–0.230.3–0.80.09±0.033GFZ 1.01–1.840.0620.5–1.8 6.4–9.6n.a.IBP 3.24–4.010.2099–2221–35>3FEP 1.18–1.670.110 3.3–5.910–14n.a.KEP 0.68–1.590.031n.a.n.a.n.a.NPX 0.39–0.930.0050.4–0.8 1.0–1.90.08±0.016DCF 0.31–0.520.010≤0.1≤0.1<0.02IDM 1.14–1.620.444≤0.21≤0.3n.a.PPZ 0.08–0.170.024n.a.n.a.n.a.CBZ0.07–0.140.048n.a.n.a.<0.005The range indicates the 95%confidence interval obtained from thebatch experiments in this study and previous research [16].The sign “±”indicates standard errors.The sign “≤”indicates that the lower limit was beyond experimental resolution [16];n.a.:data not available.aNAS:k biol for enriched nitrifier culture in this study.bCAS:k biol for conventional activated sludge [17].cMBR c :k biol for sludge taken pilot-scale membrane bioreactor [16].dCAS:k biol for sludge taken from full-scale conventional activated sludge process [16].eMBR:k biol for sludge taken from single-house membrane bioreactor at Experiment 2[18].Fig.2.Changes in NH 4-N and NO 3-N concentrations during the degradation of PhACs by CAS and NAS cultures.total mixed liquor suspended solids,and the floc size of the sludge [16,20,21].In addition the difference in the initial concentration of selected pharmaceuticals and the experimental conditions may have an influence.3.3.Degradation of PhACs by conventional activated sludgeThe elimination of pharmaceuticals by conventional activated sludge (CAS)has been repeatedly studied and those studies have shown that the removal efficiency of pharmaceuticals was gener-ally low,especially for persistent pharmaceuticals such as CA,DCF,CBZ and PPZ of which removal efficiency was around 5–20%[22].To illustrate whether there is any advantage to the NAS system compared with the CAS system in the PhACs degradation,a lab-oratory study was implemented with NAS and CAS systems under the same initial operating conditions with MLSS concentration of 1000mg/l,NH 4-N 100mg/l and without acetate as an organic sub-strate.In this study,an NAS system with nitrifying activity of 30mg NH 4-N/g MLVSS obtained from the enrichment process was used to ensure stable nitrification.The representative results on nitrifi-cation are shown in Fig.2.Although initial nitrifying activity was higher in the case of NAS than CAS,the nitrification was completed even within 6days in the cases of both NAS and CAS (Fig.2).The results on the PhACs degradation by NAS and CAS after 6days of operation are shown in Fig.3.The overall removal efficiencies for 10PhACs by the NAS system varied from 18%to 99.8%,in which 3of 10pharmaceuticals such as CA,CBZ and PPZ were removed at less than 40%efficiency (as shown in Fig.3),probably due to their lower hydrophobicity (log K ow <3)and chemical structure (Table 1).According to Kimura et al.[23]complicated chemical structures such as KEP an NPX were not removed during conventional treat-ment plants but were eliminated by membrane bioreactor [23].It was assumed that the poor removal was due to the presence of a complicated structure with two aromatic rings causing more resis-tance to the degradation process.Additionally,the presence of nitro and halogen groups in aromatic compounds probably results in a decreasing degradation rate [23,24].Our results also show that the removal efficiencies of IBP were 94%and 100%by CAS and NAS,respectively.This indicates that IBP at least as a parent compound was biodegradable with a very small difference between NAS and CAS.In contrastto the case of IBP,the removal efficiency of other pharmaceuticals such as CA,DCF,CBZ,and PPZ by CAS was very low,while NAS displayed considerable removal efficiencies.The persistence of these compounds in sewage treatment plants was repeatedly reported by several studies [16,22].The advantages of the use of nitrifiers in the degradation of persistent organic pollu-tants were reported in several recent studies [10,11,15].A detailed monitoring in a full scale plant shows that the total concentra-tion of 66pharmaceuticals in the effluent from the CAS process was 1.5times higher than that from a biological nutrient removalparison of the removal efficiency of PhACs by CAS and NAS cultures.Error bars represent the standard deviation of three triplicates.N.H.Tran et al./Journal of Hazardous Materials171 (2009) 1051–10571055Fig.4.Effect of initial ammonium concentration on the degradation of PhACs.Error bars represent the standard deviation of three replicates.process[25].The high removal under nitrification may be caused by the diversity of nitrifiers and a variety of non-specific mono-and dioxygenase enzymes associated with both heterotrophic and autotrophic microorganisms.Recent studies also found that AMO played an important role in the conversion of remarkably broad substrates including trichloroethylene,alkanes,alkenes,aromatic derivatives,several heterocyclic compounds,and some heteroatom ring compounds[10,11,13,26].Taken together,the results may suggest that the removal effi-ciency of pharmaceuticals in biological wastewater treatment not only depends on the chemical characteristics of target pharma-ceuticals but also relies on the microbial species especially on the nitrifiers involved.3.4.Effect of initial ammonium concentrations on thedegradation of PhACsTo determine the effect of ammonium concentration(as a pri-mary substrate)on the cometabolic activity of PhACs by NAS,a series of batch biodegradation experiments under the different ini-tial ammonium concentrations ranging from20to200NH4-N/l were conducted.Fig.4shows the biodegradation of100␮g/l PhACs by NAS with various initial ammonium concentrations after6days of cultivation.From Fig.4,it can be concluded that the removal efficiency of the PhACs increased with the increase in the initial ammonia concentration.In particular,the removal of persistent PhACs such as CA,DCF,CBZ,and PPZ was enhanced at higher initial ammonia concentrations.At lower initial ammonium concentra-tions,smaller cometabolic activity was probably responsible for the slower degradation of some pharmaceuticals.Although the amounts of NH4-N consumed doubled in the cases between NH4-N100mg/l and200mg/l,the removal of PhACs did not increase so much.The rate of the degradation would be dependent on the amount of AMO induced.3.5.Effect of addition of organic substrates on the degradation of PhACsGenerally speaking,autotrophs are responsible for nitrification, although heterotrophs are also known to function as nitrifiers[27]. In addition,heterotrophs can use organic compounds synthesized by autotrophs in the NAS system in this study.To clarify the role of heterotrophs,the addition of sodium acetate(a primary substrate supporting for the growth of heterotrophic bacteria contained in an NAS system)with different initial acetate concentrations ranging from0to100mg CH3COO−/l was examined without the supply of ammonium.The results are summarized in Table3and Fig.5.Fig.5shows the biodegradation efficiencies of100␮g/l PhACs by NAS after6days for the different acetate concentration addi-tions.Without acetate addition,in the case that only NAS and the selected PhACs diluted in methanol were added into MSM medium, the biodegradation of the selected PhACs such as CA,DCF,CBZ,and PPZ was not significant while IBP and FEP displayed considerable biodegradation efficiencies.In this case,methanol is considered as a carbon source supporting the growth of the heterotrophic microorganisms contained in the NAS.On the other hand,a sig-nificantly higher biodegradation efficiency of PhACs was observed in the case of acetate addition.Table3also shows the increase in the biomass concentration during the batch experiment caused by both acetate and methanol,carbon and energy sources supporting the Table3Changes in COD,and MLSS concentrations during the batch experiments of organic substrate addition.Acetate dosage(mg CH3COO−/l)Initial COD(mg/l)Final COD(mg/l) MLSS(mg/l)0108±610±2132±22 50162±88±2237±21 100209±512±3350±241056N.H.Tran et al./Journal of Hazardous Materials171 (2009) 1051–1057Fig.5.Effect of initial acetate concentration on the degradation of PhACs.Error bars represent the standard deviation of threereplicates.Fig.6.Changes in NH 4-N,NO 3-N and COD concentrations during the degradation of PhACs by NAS with and without inhibitors.growth of heterotrophic microorganisms in the enriched nitrifier culture.Urase and Kikuta [17]reported that the removal of pharmaceu-ticals was greater in the case of low organic substrate concentration in the batch experiment using conventional activated sludge.The opposite result of this study may be due to the difference in the sludge population and the amount of enzymes induced at the start of the batch experiment.In the NAS system,the amount of enzymes responsible for the degradation by heterotrophs was not enough at the start of batch experiments,because the MSM used in the enrichment period did not include any organic substrate.Conse-quently in the NAS system,the degradation of PhACs was promoted by the organic substrates by the induction of the enzymes.On the other hand,in the CAS system,PhACs could compete with organic substrates in the degradation because the enzymes had already been induced at the start of the batch experiments.Although the amount of enzymes induced before the start of the batch exper-iment affected the results,there are several researches showing the increase in the removal efficiency of micropollutants with higher organic substrate concentrations.Alexy et al.[28]foundthat partial biodegradation of 18biologically persistent antibiotics was observed when sodium acetate was also present.Ziagova and Liakopoulou-Kyriakides [29]reported that the degradation of 2,4-dichlorophenol was enhanced by the addition of glucose.3.6.Degradation of PhACs by enriched nitrifier culture in the presence of inhibitorsTo reinforce the contributive role of the nitrification process in the degradation of PhACs by NAS,two inhibitory reagents were used to confirm whether the removal of pharmaceuticals arose from the biological activity of sludge,particularly from the activity of ammo-nia oxidizing bacteria (AOB)community.ATU and NaN 3were used to inhibit ammonium oxidation activity and the complete biological activity of sludge,respectively.As shown in Fig.6,without inhibitors,the ammonium in the solution was converted to nitrate,and the total nitrogen con-centrations were nearly constant.In contrast,in the case of ATU addition,the nitrification was negligible however,COD concen-tration decreased significantly from 102to 8mg/l duringtheFig.7.Removal of the PhACs by NAS with and without inhibitors.Error bars represent the standard deviation of three triplicates.N.H.Tran et al./Journal of Hazardous Materials171 (2009) 1051–10571057experimental period due to the degradation of methanol added as the solvent for the pharmaceuticals.The differences between the results of the experiments with and without ATU addition in Fig.7 can be interpreted as being due to biodegradation by nitrification. Because ATU is only an inhibitor of ammonia monooxygenase in ammonia oxidizing bacteria,the high degradation of IBP and par-tial degradation of other selected PhACs such as GFZ,KEP,FEP,NPX, IDM and DCF were observed during the degradation period prob-ably due to the activity of heterotrophs.The degradation of IBP in the enriched nitrifier culture was mainly dependent on the activity of heterotrophic microorganisms.That is why CAS in the previous study[16]can degrade IBP much faster than NAS in this study.In the case of NaN3addition,the changes in pharmaceuti-cals concentrations were not significant.Sodium azide inactivates almost all of the biological activity of microorganisms,and thus the removal of the pharmaceuticals in the experiment with NaN3was caused solely by adsorption onto sludgeflocs without biological degradation.Although sodium azide may alter the surface char-acteristics/adsorptive capacity of sludgeflocs,the low adsorption of the pharmaceuticals onto sludge in the batch experiment was consistent with the previous study[17].4.ConclusionThe biodegradation of10selected pharmaceuticals by enriched nitrifier cultures with ammonia oxidizing activity of30mg NH4-N/g MLVSS h was investigated under various operating conditions. Obvious advantages of the enriched culture of nitrifier over the conventional activated sludge were recognized with most of the selected pharmaceutical substances tested.In particular,the per-sistent pharmaceuticals such as DCF,NPX,CBZ and PPZ were removed by enriched nitrifier culture more efficiently than by the conventional activated sludge.The experimental results have demonstrated that the high removal efficiency of pharmaceuti-cals was observed under the supplement of growth substrates (ammonium and organic substrates)probably depending on the amount of enzymes induced.The addition of ATU,an ammonia monooxygenase inhibitor,inhibited the nitrification completely and suppressed the removal of most selected pharmaceuticals.The partial biotransformation of ibuprofen and other selected PhACs in the presence of ATU may be due to the activity of heterotrophic microorganisms in the enriched nitrifier culture.In conclusion,the results taken together suggest that the elimination of selected pharmaceuticals can be enhanced by nitri-fication.The enrichment of nitrifier-dominated culture to achieve stable nitrification in the activated sludge may have an advantage in enhancing the removal efficiency of organic micropollutants. 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The Principles and Properties ofBiophysicsBiophysics is a field that seeks to understand the physical principles that underlie the behavior of biological systems. It is an interdisciplinary field that combines physics, biology, and chemistry to study the properties of living things. Biophysics is concerned with the structure and function of biological molecules, the mechanics of cells and tissues, and the interactions between biological systems and their environment.The principles of biophysics are rooted in physics. At the most fundamental level, biophysics seeks to understand the behavior of biological systems in terms of the laws of physics. This means that biophysicists must have a deep understanding of physics, including mechanics, thermodynamics, electromagnetism, and quantum mechanics. They use this knowledge to develop models and theories that can explain the behavior of living things.One of the key properties of biological systems is complexity. Living things are made up of a vast array of molecules and structures, all of which interact with each other in complex ways. Biophysics seeks to understand this complexity by studying the properties of individual molecules and structures, and how they interact with each otherto form larger systems.One of the most important tools that biophysicists use to study biological systems is imaging. Advances in imaging technology have allowed researchers to observe biological systems at increasingly high resolutions, from the level of individual molecules to entire organisms. Techniques like X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and fluorescence microscopy have revolutionized our understanding of the structure and function of biological systems.Another key property of biological systems is the ability to adapt and change in response to their environment. Biophysicists study how living things respond to changes in their environment, such as changes in temperature, pressure, or pH. They are alsointerested in how living things sense and respond to different stimuli, such as light, sound, and chemicals.One of the most exciting areas of biophysics research is the study of biomaterials. Biomaterials are materials that are designed to interact with biological systems, either to support and enhance their function, or to replace damaged or diseased tissues. Biophysicists are developing new materials that can mimic the properties of biological tissues, such as skin, muscle, and bone, with the goal of creating new medical treatments and therapies.Biophysics is an interdisciplinary field that is essential for understanding the properties and behavior of biological systems. By combining physics, biology, and chemistry, biophysicists are shedding new light on the complex and fascinating world of living things. As imaging technology and computational methods continue to advance, the potential for new discoveries in biophysics is enormous.。

生物识别技术外文翻译文献生物识别技术外文翻译文献(文档含中英文对照即英文原文和中文翻译)外文：The first chapter1.1 The research background of iris recognitionBiometrics is a technology for personal identification using physiological characteristics and behavior characteristics inherent in the human body. Can be used for the biological characteristics of biological recognition, fingerprint, hand type face, iris, retina, pulse, ear etc.. Behavior has the following characteristics: signature, voice, gait, etc.. Based on these characteristics, it has been the development of hand shape recognition, fingerprint recognition, facial recognition, iris recognition, signature recognition and other biometric technology, many techniques have been formed and mature to application of.Biological recognition technology in a , has a long history, the ancient Egyptians through identification of each part of the body size measure to carry out identity may be the earliest human based on the earliest history of biometrics. But the modern biological recognition technology began in twentieth Century 70 time metaphase, as biometric devices early is relatively expensive, so only a higher security level atomic test, production base.due to declining cost of microprocessor and various electronic components, precision gradually improve, control device of a biological recognition technology has been gradually applied to commerce authorized, such as access control, attendance management, management system, safety certification field etc..All biometric technology, iris recognition is currently used as a convenient and accurate.Making twenty-first Century is information technology, network technology of the century, is also the human get rid of traditional technology, more and more freedom of the century. In the information, free for the characteristics of the century, biometric authentication technology, high-tech as the end of the twentieth Century began to flourish, will play a more and more important role in social life, fundamentally change the human way of life . Characteristics of the iris, fingerprint, DNA the body itself, will gradually existing password, key, become people lifestyle, instead of at the same time, personal data to ensure maximum safety, maximize the prevention of various types of crime, economic crime.Iris recognition technology, because of its unique in terms of acquisition, accuracy and other advantages, will become the mainstream of biometric authentication technology in the future society. Application of safety control, the customs import and export inspection, e-commerce and other fields in the future, is also inevitable in iris recognition technology as the focus. This trend, now in various applications around the world began to appear in the.1.2 Objective and significance of iris recognitionIris recognition technology rising in recent years, because of its strong advantages and potential commercial value, driven by some international companies and institutions have invested a lot of manpower, financial resources and energy research. The concept of automatic iris identification is first proposed by Frown, then Daugman for the first time in the algorithm becomes feasible.The iris is a colored ring in the pupil in the eye of fabric shape, each iris contains a structure like the one and only based on the crown, crystalline, filaments, spots, structure, concave point, ray, wrinkles and fringe characteristic. The iris is different from the retina, retinal is located in the fundus, difficult to image, iris can be seen directly, biometric identification technology can obtain the image of iris fine with camera equipment based on the following basis: Iris fibrous tissue details is rich and complicated, and the formation and embryonic tissue of iris details the occurrence stage of the environment, have great random the. The inherent characteristics of iris tissue is differ from man to man, even identical twins, there is no real possibility of characteristics of the same.When the iris are fully developed, he changes in people's life and tiny. In the iris outer, with a layer of transparent corneal it is separated from the outside world. So mature iris less susceptible to external damage and change.These characteristics of the iris has the advantages, the iris image acquisition, the human eye is not in direct contact with CCD, CMOS and other light sensor, uses a non technology acquisition invasion. So, as an important biometric identity verification system, iris recognition by virtue of the iris texture information, stability, uniqueness and non aggressive, more and more attention from both academic and industrial circles.1.3 Status and application of domestic and foreign research on iris recognitionIDC (International Data Group) statistics show that: by the end of 2003, the global iris recognition technology and related products market capacity will reach the level of $2000000000. Predicted conserved survey China biometric authentication center: in the next 5 years, only in the Chinese, iris recognition in the market amounted to 4000000000 rmb. With the expansion of application of the iris recognition technology, and the application in the electronic commerce domain, this number will expand to hundreds of billions.The development of iris recognition can be traced back to nineteenth Century 80's.. In 1885, ALPHONSE BERTILLON will use the criminal prison thoughts of the application of biometrics individual in Paris, including biological characteristics for use at the time: the size of the ears, feet in length, iris.In 1987, ARAN SAFIR and LEONARD FLOM Department of Ophthalmology experts first proposed the concept, the use of automatic iris recognition iris image in 1991, USA Los ala Moss National Laboratory JOHNSON realized an automatic iris recognition system.In 1993, JOHN DAUGMAN to achieve a high performance automatic iris recognition system.In 1997, the first patent Chinese iris recognition is approved, the applicant, Wang Jiesheng.In 2005, the Chinese Academy of Sciences Institute of automation, National Laboratory of pattern recognition, because of outstanding achievement "in recognition of" iris image acquisition and aspects, won the two "National Technology Invention Prize", the highest level represents the development of iris recognition technology in china.In 2007 November, "requirements for information security technology in iris recognition system" (GB/T20979-2007) national standards promulgated and implemented, the drafting unit: Beijing arithen Information Technology Co., ltd..Application of safety control, the customs import and export inspection, e-commerce and other fields in the future, is also inevitable in iris recognition technology as the focus. This trend, now in various applications around the world began to appear in the. In foreign countries, iris recognition products have been applied in a wide range.In February 8, 2002, the British Heathrow Airport began to test an advanced security system, the new system can scan the passenger's eyes, instead of to check passports. It is reported, the pilot scheme for a period of five months, a British Airways and virgin Airlines passengers can participate in this test. The International Air Transport Association interested in the results of this study are, they encourage the Heathrow Airport to test, through the iris boarding passengers to determine its identity as a boarding pass.Iris recognition system America "Iriscan" developed has been applied in the three business department of Union Bank of American Texas within. Depositors to be left with nothing whatsoever to banking, no bank card password, no more memories trouble. They get money from the A TM, a camera first eye of the user to scan, and then scan the image into digital information and data check, check the user's identity.America Plumsted school in New Jersey has been in the campus installed device of iris recognition for security control of any school, students and staff are no longer use cards and certificates of any kind, as long as they passed in the iris camera before, their location, identity is system identification, all foreign workers must be iris data logging to enter the campus. At thesame time, through the central login and access control system to carry on the control to enter the scope of activities. After the installation of the system, various campus in violation of rules and infringement, criminal activity is greatly reduced, greatly reducing the campus management difficulty.In Afghanistan, the United Nations (UN) and the United Nations USA federal agency refugee agency (UNHCR) using iris recognition system identification of refugees, to prevent the same refugee multiple receive relief goods. Use the same system in refugee camps in Pakistan and Afghanistan. A total of more than 2000000 refugees use iris recognition system, this system to a key role for the United Nations for distribution of humanitarian aid from.In March 18, 2003, Abu Zabi (one of the Arabia and the United Arab Emirates) announced the iris recognition technology for expelled foreigners iris tracking and control system based on the borders opened the world's first set of national level, this system began construction from 2001, its purpose is to prevent all expelled by Abu Zabi tourists and other personnel to enter the Abu Zabi. Without this system in the past, due to the unique characteristics of the surface of the Arabs (Hu Xuduo), and the number of the expulsion of the numerous, customs inspection staff is very difficult to distinguish between what is a deported person. By using this system, illegal immigration, all be avoided, the maximum guarantee of national security.Kennedy International Airport in New Jersey state (John F. Kennedy International Airport) of the iris recognition system installed on its international flights fourth boarding port, 300 of all 1300 employees have already started to use the system login control. By using this system, all can enter to the apron personnel must be after the system safety certification of personnel. Unauthorized want to break through, the system will automatically take emergency measures to try to force through personnel closed in the guard space. Using this system, the safety grade Kennedy International Airport rose from B+ to A+ grade. The Kennedy International Airport to travel to other parts of the passengers has increased by 18.7%.Generally speaking, the iris recognition technology has already begun in all walks of life in various forms of application in the world. At the same time, to the application of their units of all had seen and what sorts of social benefits and economic benefits are not see. This trend is to enhance the high speed, the next 10 years will be gradually achieve the comprehensive application of iris recognition in each industry.In China, due to the Chinese embargo and iris technology itself and the difficulty in domestic cannot develop products. So far, there has not been a real application of iris recognition system. However, many domestic units are expressed using strong intention, especially the "9 · 11" later, security anti terrorism consciousness has become the most concerned problems in the field of aviation, finance. Iris recognition system is a major airline companies, major financial institutions and other security mechanisms (such as aerospace bureau) become the focus of attention of object and other key national security agency. As with the trend of development in the world, iris recognition technology will in the near future in application China set off climax.The second chapter of introduction of iris recognition technology2.1 Technology of biological feature recognition based on2.1.1 Present status and development of biological feature recognition“9.11" event is an important turning point in the development of biometric ident ification technology in the world, the importance of it makes governments more clearly aware of the biological recognition technology. Traditional identity recognition technologies in the face ofdefect anti terrorism has shown, the government began a large-scale investment in the research and application of biometric technology. At the same time, the public understanding of biological recognition technology with "9.11" exposure rate and greatly improve the.The traditional method of individual identification is the identity of the people with knowledge, identity objects recognition. The so-called identity: knowledge refers to the knowledge and memory system of personal identification, cannot be stolen, and the system is easy to install, but once the identification knowledge stolen or forgotten, the identity of easily being fake or replaced, this method at present in a wide range of applications. For example: the user name and password. The so-called identity items: refers to the person, master items. Although it is stable and reliable, but mainly depend on the outer body, lost or stolen identification items once proof of identity, the identity of easily being fake or replaced, for example: keys, certificates, magnetic card, IC card etc..Biometric identification technology is related to physical characteristics, someone using prior record of behavior, to confirm whether the facts. Biometric identification technology can be widely used in all fields of society. For example: a customer came into the bank, he did not take bank card, also did not remember the password directly drawing, when he was drawing in the drawing machine, a camera to scan on his eyes, and then quickly and accurately complete the user identification and deal with business. This is the application of the iris recognition system of modern biological identification technology. "".America "9.11" after the incident, the anti terrorist activity has become the consensus of governments, it is very important to strengthen the security and defense security at the airport, some airports USA can in the crowd out a face, whether he Is it right? Wanted. This is the application of modern technology in biological feature recognition "facial recognition technology".Compared with the traditional means of identity recognition, biometric identity recognition technology in general has the following advantages:(1) the security performance is good, not easy to counterfeit or stolen.(2) carry, whenever and wherever possible, therefore more safety and security and other identification method.For the biological information of biometric recognition, its basic nature must meet the following three conditions: universality, uniqueness and permanency.The so-called universality, refers to any individual has the. Uniqueness, is in addition to other than himself, other people did not have any, namely different. The so-called permanent, refers to the character does not change over time, namely, life-long.Feature selection of organisms with more than three properties, is the first step of biological recognition.In addition, there are two important indexes in biological recognition technology. The rejection rate and recognition rate. Adjusting the relation of these two values is very important. The reject rate, the so-called false rejection, this value is high, use frequency is low, the error recognition, its value is high, safety is relatively reduced. So in the biological identification of any adjustment, the two index is a can not abandon the process. The choice of range size, related to the biological identification is feasible and available .And technology of identity recognition based on iris feature now appears, it is the development of biometric identification technology quickly, due to its uniqueness, stability,convenience and reliability, so the formation of biometric identification technology has the prospects for development.Generally speaking, the biological recognition system consists of 4 steps. The first step, the image acquisition system of collecting biometric image; the second step, the biological characteristics of image preprocessing (location, normalization, image enhancement and so on); the third step, feature information extraction, converted into digital code; the fourth step, the generation of test code and database template code to compare, make identification。

Enzyme and Microbial Technology41(2007)570–575Characteristic of␤-glucosidase from Sicilian bloodoranges in relation to anthocyanin degradationR.N.Barbagallo a,∗,R.Palmeri a,S.Fabiano a,P.Rapisarda b,G.Spagna a,∗a Dipartimento di OrtofloroArboricoltura e Tecnologie Agroalimentari(DOFATA),Via Santa Sofia98,951123Catania,Italyb CRA-Istituto Sperimentale per l’Agrumicoltura,Corso Savoia190,95024Acireale(CT),ItalyReceived5August2005;received in revised form9May2007;accepted9May2007AbstractAnthocyanin content in Sicilian sweet orange[Citrus sinensis(L)Osbeck]varieties known as blood oranges(Tarocco,Moro e Sanguinello) undergoes changes during the ripening process.Concentration increases during ripening,reaching a maximum in the fully ripe fruit.At latter stage of maturity,a decrease of these pigments is observed.This study aims to evaluate␤-d-glucosidase activity(␤G,EC3.2.1.21)in Tarocco variety, the most common Sicilian blood orange,and to determine the main physicochemical and kinetic properties of this enzyme in order to underline its role on anthocyanins degradation during ripening.The enzymatic extract was assayed on both anthocyanins extract from Tarocco juice and a synthetic substrate(p-nitrophenyl-␤-d-glucopyranoside,p NPG).It was observed a400times higher specificity of the synthetic substrate than the natural substrate.Kinetic studies and physicochemical characterization of␤G such as V max and K m(V max2.1×10−2and K m2.67×10−4using p NPG as substrate;V max3.3×10−3and K m2.1×10−1using natural substrate),optimum conditions of pH(4.5)and temperature(60◦C),fructose and glucose inhibition(competitive inhibition by glucose)and thermal stability(decimal reduction time3min at75◦C and decimal reduction temperature9.5◦C)were also performed.␤-d-Glucosidase activity seems involved on anthocyanins degradation duringfinal ripening stage of fruit.©2007Elsevier Inc.All rights reserved.Keywords:Tarocco orange;␤-d-Glucosidase;Anthocyanins;Stability;Ripening1.IntroductionCitrus fruit are important sources of health promoting con-stituents.In addition to ascorbic acid,these includeflavonoids, hydroxycinnamic acids,carotenoids and limonoids[1].Some of these compounds are present as glycosides and the type and position of glycosidic substituent has a profound effect on taste,solubility and bioavailability.Anthocyanins are glycosides belonging to theflavonoid compounds,a class of plant secondary metabolites responsible for the rind andflesh red color of some Sicilian sweet orange[Citrus sinensis(L)Osbeck]varieties known as blood oranges(Tarocco,Moro e Sanguinello).Antho-cyanin content has been considered as an important quality attribute in both fresh fruit market and processing industry due to its biological activity[2,3].Several anthocyanins have been ∗Corresponding author at:Food Biotechnology Group,DOFATA,University of Catania,Via S.Sofia98,95123Catania,Italy.Tel.:+390957580213; fax:+390957141960.E-mail addresses:rbarbaga@unict.it(R.N.Barbagallo),gspagna@unict.it(G.Spagna).identified in blood oranges juice,with cyanidin-3-glucoside and cyanidin-3-(6 -malonyl)-glucoside as the major pigments [4,5].Anthocyanins stability in fruit is promoted by copigmenta-tion with otherflavonoids or phenolic acids,self-association, and low pH[6].On the other hand,enzymatic systems present in plant tissues such as anthocyanase(anthocyanin-␤-glucosidase) and polyphenoloxidase could play an important role in degrada-tion process of anthocyanins.Such enzymes mainly act during senescence of fruit,hydrolising and oxidizing the anthocyanin molecules.Few works have been carried out on citrus fruit␤-glycosidase(␤G).Burns[7]found a low level of␤-glycosidase activity in sweet orange fruit juice vesicles,while no activity of this enzyme was detected in grapefruit peel and juice vesicle [8–10].Finally,a recent study[11]put in evidence the pres-ence of␤G in fruitflavedo and albedo tissue of Valencia orange variety.Despite the importance of␤-d-glucosidase(␤G,EC 3.2.1.21)activity onflavour profile of many fruit,few researches were found about its role on anthocyanins degradation.Due to loss of glycosidic moiety for hydrolysis,the aglycon(antho-cyanidin)could become extremely unstable with consequent0141-0229/$–see front matter©2007Elsevier Inc.All rights reserved. doi:10.1016/j.enzmictec.2007.05.006R.N.Barbagallo et al./Enzyme and Microbial Technology41(2007)570–575571 adverse effect on juice color and represents a good substratefor other enzymes.The present study aims to evaluate␤-d-glucosidase activityin Tarocco orange and to determine the main physicochemicaland kinetic properties of such enzyme in order to underline itsrole on the anthocyanins degradation during different ripeningstages.2.Materials and methods2.1.Plant materialsThe study was performed on fruit from Tarocco cultivar(“Arcimusa”clone)hand-harvested randomly fromfive trees,grafted on sour orange(C.aurantiumL.),and planted at the“Palazzelli”(Siracusa,Italy)experimental farm of theCRA–Istituto Sperimentale per l’Agrumicoltura of Acireale(Catania,Italy).Twenty fruit from each tree were collected atfive different ripening stages,from February to May,2005.Each fruit was squeezed separately using ahousehold juicer(Moulinex,Milan,Italy)and the cloudy juice obtained wascentrifuged at20,000×g for30min at5◦C,in order to separate the residualpulp from the juice.These fractions were stored singularly at−50◦C prior tophysicochemical and enzymatic analysis.2.2.Physicochemical analysesTotal soluble solid(TSS),total acidity(TA),ratio(TSS/TA)were determinedon cloudy juice according to standard methods[12].Ascorbic acid content wasdetermined on centrifuged juice by2,6-dichlorophenolindophenol titrimetricmethod modified by Rapisarda and Intelisano[13].Total anthocyanins in juice(a)and extract(b)were determined spectrophotometrically by pH differentialmethod[14,15].For determination in juice(a)an aliquot of juice(2mL)wasdiluted up to25mL with a pH1solution(125mL of0.2M KCl and375mLof0.2M HCl).A second aliquot(2mL)was diluted up to25mL with a pH4.5buffered solution(400mL of1M CH3CO2Na,240mL of1M HCl,and360mLof H2O).Absorbance of the solutions was measured at510nm.Concentrationof anthocyanins was calculated by the equation:C mg/L=(Abs pH1−Abs pH4.5)×484.82×1000 24,825×DFwhere the term in parentheses is the difference of absorbance at510nm between pH1and pH4.5solutions,484.82is the molecular mass of cyanidin-3-glucoside chloride,24,825is its molar absorptivity(ε)at510nm in the pH1solution,and DF is the dilution factor.Accuracy of results by this method is supported by independent HPLC data.For anthocyanins extraction(b),3L of Tarocco orange juice previously centrifuged were loaded on a glass column(length40cm;i.d. 2cm)filled with a nonpolar SDVB(styrene-divinylbenzene)resin Kastel S-112(Dow-Italia,Milano,Italy).After the adsorption,resin was washed with 2–3volumes of distillated water in order to obtain sugars and other soluble components elution.Anthocyanins were desorbed using1%HCl methanol(v/v) and different fraction were collected.Methanolic solution of the anthocyanins was concentrated by rotary evaporator maintaining the temperature below35◦C. This extract was used for enzymatic assay.The juice color was determined with a compact tristimulus chromameter (Minolta CR-300,Ramsey,NJ,USA)with anØ8mm viewing aperture,white plate reference(Y=94.3;x=0.3142;y=0.3211)and C illuminant(CIE,2◦observer)was used.Readings were expressed as L*,a*and b*parameters. Chroma[(a*2+b*2)1/2]and Hue angle[tan−1(b*/a*)]were calculated.2.3.Enzymes determinationAn aliquot of pulp(50g)was homogenised at4.0◦C by adding200mL of extracting solution(citrate–phosphate buffer,0.1M;NaCl,1M;dithiothreitol, 1mM;pH5.0)[16].Suspension was maintained under stirring for2h at4.0◦C, then centrifuged at10,000×g for15min at4◦C.Starting from the extraction at optimum pH conditions,surfactants PEG400and PEG4000were tested by dissolving in1/3part of the extracting solution volume and adding to the suspension with the other2/3parts of the solution before pH correction.Tested concentrations of both surfactants were up to1.5%(w/v);besides,concentrations tested for PEG4000were2.3and4.0%(w/v).In order to maximize yield,the extract was:(a)saturated with ammonium sulphate80%,kept at4.0◦C overnight andfiltrate under vacuum with a5892filter(Whatman);(b)directlyfiltrate and concentrated by tangentialflow ultrafiltration with50kDa cut-off membrane, and retentate utilised as enzymatic extract.The juice was directly analysed.␤G activity was determined by hydrolysis of synthetic(p-nitrophenyl-␤-d-glucopyranoside,p NPG)and natural substrates(orange anthocyanins extract) at pH4.0and30◦C[17].An aliquot of0.05mL of diluted enzyme was added to0.45mL of0.55×10−2M p NPG dissolved in0.1M citrate–phosphate(C–P) buffer pH5.0at25◦C under continuous stirring.After1min,reaction was stopped by adding1mL sodium carbonate1M.Blank was carried out by invert-ing sodium carbonate with the enzyme solution.The absorbance of released 4-nitrophenolate ion was read at400nm and the concentration determined by usingε18,300M−1cm−1.One unit of enzyme activity was defined as the amount of enzyme releasing1␮mol of4-nitrophenol min−1under assay conditions.For anthocyanase determination[18]was used the cyanidin-3-glucoside extinction coefficient(ε=24,825M−1cm−1)at510nm.Enzymatic units were defined as micromoles of anthocyanins.Polyphenoloxidase(PPO,EC1.14.18.1)was assayed spectrophotometri-cally at505nm using3,4-dihydroxy phenylacetic acid(DOPAC)as phenolic substrate with MBTH(3-methyl-benzothyazolinone hydrazone)to trap the enzyme-generated ortoquinone[19,20].The absorbance was read at505nm and concentration determined by usingε25,400M−1cm−1.One unit of PPO activity was defined as the amount of enzyme which produces1␮mol of adduct for min at25◦C.Protein concentration was determined after precipitation in7%(w/v) trichloroacetic acid by means of Coomassie Blue G250and by employing BSA as standard[21].The solvents and reagents not expressly specified had a high degree of purity(RPE)and were supplied by Carlo Erba(Rodano,Italy).2.4.Characterization ofβGThe following physicochemical and kinetic parameters were established: optimum temperature,optimum pH,inhibitions(glucose,fructose,ethanol), V max,K m,thermo-stability and molecular weight by SDS-PAGE.2.4.1.Optimum temperatureThe enzyme was assayed at temperatures from21to90◦C(at pH5.0),with 600mL of1M sodium carbonate added in order to stop the reaction.2.4.2.Optimum pHCitric–citrate buffer solutions(0.1M)were prepared under different pH con-ditions(from2.5to8.0at30◦C)in such a way as to assess the enzyme behaviour even in very different conditions to those of orange juices.2.4.3.Glucose and fructose inhibitionsGlucose and fructose inhibition tests were performed using sugar concen-trations not exceeding25g L−1(at pH5.0and30◦C).The enzyme assay was carried out as previously described except as regards the sugar and buffer con-centrations,added in proportional amounts with respect to thefinal volume of the assay.2.4.4.Kinetic parameters(V max and K m)The behaviour of the enzyme(at pH5.0and30◦C)was explained by the Michaelis–Menten equation and the kinetic parameters calculated by way of graphical extrapolation according to Lineweaver–Burk[22].Substrates were prepared increasing concentration until saturation.2.4.5.StabilityThree aliquots of extract(60mL)were lyophilized and dissolved in10mL citric–citrate buffer0.1M pH5.0.Three tests were prepared by adding0.50␮L to1.5mL of pasteurized(90◦C for30min)and centrifuged orange juice;the three samples were put in thermostat at temperatures of37,52and70◦C.572R.N.Barbagallo et al./Enzyme and Microbial Technology 41(2007)570–5752.4.6.SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis)The enzyme extract was performed as described by Laemmhi [23].The samples,previously partially purified by purified by ammonium sulphate pre-cipitation (80%saturation)were put in touch with a 12%(w/v)polyacrylamide gel,both were dissolved according to the protein content determined by the above cited Coomassie Blue G250and by employing BSA as standard [21].2.5.Statistical analysisAll determinations were conducted three times at least.Analysis of variance (ANOV A)of the data was evaluated by the Statistical Analysis System (SAS version 9.0).Duncan’s Multiple Range Test was employed to determine the statistical significance of the differences between the means (p ≤0.05).3.Results and discussion 3.1.Physicochemical analysesVariations of physicochemical parameters during orange mat-uration process were reported in Table 1.Total soluble solids (TSS)showed a slight increase between February and first days of May;after this period a decrease was noticed.Total acidity (TA)decreased quickly until 14March and then remained quite constant.The ratio (TSS/TA)values revealed that in March sam-ples reached the optimal maturity stage.Besides,although it was noticed an increasing of TSS and TA values at first sampling of May,ratio value remained unchanged.Ascorbic acid content was stable during maturation except than in the last stage in which a decrease was observed.The anthocyanin content reached a max-imum in 14March when fruit were fully ripe.After this stage,values decreased.That was the most evident result between last two sampling,in which biochemical senescence process is more active.In samples withdrawn on 24May,anthocyanin content reduced to half in comparison with the maximum value.L *a *b *values described changes in juice color at different stages of Sicilian blood oranges.Apart from a slight loss in lightness (L *)and green-red (a *)values after 14March,juice did not show any other significant variation of colour parameters,whereas the blue-yellow (b *)chromatism did not influence significantly the organoleptic profile.Changes in total anthocyanin amount of blood orange juice did not seem to reflect such visible changes in juice colour,since the maturation stages probably influenced stability of anthocyanins,as reported by other authors [24,25].3.2.Enzymes determinationThe analysis of raw juice showed a ␤G activity of 1.5×10−3U g −1.Relatively to pulp,extraction tests at different pH conditions were performed (Fig.1).The maximum value of enzyme activity was noticed at pH 6.0,subsequently chosen for all extractions.A higher pH cause an increasing of dissociation rate of pulp pectins carboxylic groups;it causes an increasing of the intra and inter-molecular electrostatic repulsions between the pectin chains and,probably,even between enzyme and pectin,resulting in an easier ␤G release in extraction solutions.On residual part of the first extraction,constituted by pulp and a gelatinous layer (calcium pectate),a second extraction was car-T a b l e 1P h y s i c o -c h e m i c a l c h a r a c t e r i s t i c s o f b l o o d o r a n g e j u i c e h a r v e s t e d a t fiv e d i f f e r e n t r i p e n i n g s t a g e sp HT S S (%)T A (%)T S S /T AA s c o r b i c a c i d (m g %)A n t h o c y a n i n s (m g /L )L *a *b *8F e b r u a r y 3.44±0.02a 11.61±0.2a 1.35±0.1a 8.60±0.7a 66.48±1.4b 89.70±11.35a 37.37±3.72a 23.89±0.58a 41.68±1.40a 14M a r c h 3.46±0.05a 11.98±0.5a 1.02±0.2a 11.75±1.6b 66.93±1.8b 156.00±12.90c 38.05±2.19a 24.76±0.19a 40.86±0.91a 11A p r i l 3.48±0.04a 12.00±0.5a 1.09±0.1a 11.01±0.8a b 65.24±0.9b 114.56±10.29b 35.31±1.03b 29.36±0.28b 37.18±2.33a 4M a y 3.48±0.02a 12.35±0.4a 1.19±0.2a 10.38±1.3a b 68.09±2.8b 79.63±7.15a 31.31±1.03b 31.36±0.28b 36.18±2.63a 24M a y 3.41±0.05a 11.13±0.5a 1.09±0.1a 10.21±0.8a b 48.46±6.7a 75.01±6.95a 29.31±1.03b31.30±0.26b 36.10±2.35a N o t e :M e a n o f s e p a r a t e 20f r u i t j u i c e s d e t e r m i n a t i o n .M e a n s i n a s a m e c o l u m n f o l l o w e d b y t h e s a m e l e t t e r a r e n o t s i g n i fic a n t l y d i f f e r e n t a t t h e p ≤0.05l e v e l a c c o r d i n g t o D u n c a n ’s m u l t i p l e r a n g e t e s t .R.N.Barbagallo et al./Enzyme and Microbial Technology 41(2007)570–575573Fig.1.Effect of pH extraction on ␤G activity.ried out,that led to a limited activity recover (lower than 5%).Total pulp activity resulted 8–10times higher,for all samples,than the centrifuged juice activity,since the ␤G is mostly bond to the solid fraction of fruit and,presumably,to pectin chains.In order to increase the ␤G extraction,it was tried to add a non ionic surfactant,polyethylene glycol (PEG)at two different molecu-lar weights (400and 4000),as suggested by Gunata [16]for ␤G extraction from grapes skin.The results showed a worsening in extraction yield with rela-tive specific activity of ␤G extracted of 79.74and 64.26%,with PEG 400and 4000surfactants,respectively.Probably,the dif-ferent behaviour depends to the presence of a wax layer over the grapes and/or to different molecular structure of the enzyme.The use of ammonium sulphate precipitation (80%saturation)reduced the activity to less than 1/3,so ultrafiltration was pre-ferred since its higher yield (>80%).Fig.2reports anthocyanins and ␤G changes in fruit,from February to May 2005.Anthocyanin concentrations in fruit reached a maximum at day 34and then decreased,while ␤G showed an opposite trend,proportionally increasing its activity,with a 80%correlation coefficient (95%confidence interval).Then,it would seem that ␤G plays a role in anthocyanins degra-dation of Tarocco fruit at the end of ripening.In fact,loss of glucosidic substituent from anthocyanins by ␤G hydrolisis destabilizes the anthocyanins leading to adverse effects on color quality,as the aglycon is extremely unstable.The anthocyanidins may be a good substrate for oxidases such as polyphenoloxidase (PPO)in a successive enzymatic degradation process [26].PPO activity was found in Tarocco orange (with a range fromFebru-Fig.2.Anthocyanins content and ␤-glucosidase activity during fruitripening.Fig.3.Enzyme kinetics with synthetic substrate (p NPG,3a)and natural sub-strate (anthocyanins,3b).ary to May of 2.50–2.99×10−4Ug −1),although with lower value than ␤G (ca.1/30).Presence of the PPO activity in blood orange indicates that this enzyme could play a role on antho-cyanidin degradation.3.3.Characterization of βGThe enzyme involved in p NPG hydrolysis followed a Michaelis–Menten kinetics (Fig.3a),with K m 2.67×10−4M and V max di 2.1×10−2nmol/mg proteins min.With regard to the hydrolysis of anthocyanins obtained from Tarocco extract (Fig.3b),it was observed a six times reduc-tion of enzymatic activity and enzyme inhibition at high anthocyanins concentration (product inhibition).The enzyme behaviour on anthocyanins substrate cannot be explained by Michaelis–Menten equation.Referring to the point of maximum,apparent K m was 2.1×10−1M and V max was 3.3×10−3nmol/mg proteins min.Therefore,␤G specificity on synthetic substrate compared to anthocyanins,calculated from V max /K m ratios of respective substrates,resulted 400times higher.Relative activity reached maximum value at pH 4.5(Fig.4),with a 35%of relative activity at physiological pH of the fruit (3.5).This course resulted similar in ␤G from microbial sources,as Aspergillus niger [27,28].Therefore,it could be supposed that the reaction mechanism is always an acid–base reaction with involvement of at least two carboxylic groups [28].Optimum temperature (Fig.5)resulted quite high for a vegetable source574R.N.Barbagallo et al./Enzyme and Microbial Technology 41(2007)570–575Fig.4.Effect of pH on ␤-glucosidase relativeactivity.Fig.5.Effect of temperature on ␤-glucosidase relative activity.enzyme (60◦C)and even in this case similar to enzyme from microbial source [29].The enzyme was not inhibited by fructose and ethanol,while glucose inhibition was noticed.Kinetic studybyFig.6.Glucose inhibition according to Lineweaver–Burk (a);thermal stability of ␤-glucosidase(b).Fig.7.Arrhenius plot for heat inactivation of ␤-glucosidase.Lineaweaver–Burk interpolation,allowed to establish that the inhibition was competitive,with an inhibition constant value K i ,equal to 3.08×10−2M (Fig.6a).␤G stability tests allowed to determine,by activity versus time plot (Fig.6b),the enzyme inactivation constants (k in )at various temperatures.By these results,an inactivation energy of 30kcal/mol K was calculated by the Arrhenius plot (Fig.7).Such results,expressed as second Bigelow law (ln D1/D2= T /z ),allowed to determine for ␤G a decimal reduction time (D )of about 3min at 75◦C and a deci-mal reduction temperature (z )of about 9.5◦C.Then,the enzyme showed a good stability so it could play a role in thermal process of blood orange juice.SDS-polyacrylamide gel electrophoresis of enzyme partially purified by ammonium sulphate precipitation (80%saturation)was performed as shown in Fig.8.There are two major bands corresponding to the molecular weight of 65and 55kDa.In conclusion,results obtained put in evidence for the first time presence of ␤G in blood orange cv.Tarocco .The enzyme activity in pulp resulted higher than in centrifuged juice,showing that ␤G is mainly bond to the solid part of fruit and probably to pectin chains.Specificity resulted higher for syntheticsubstrateFig.8.SDS-polyacrylamide gel electrophoresis of pulp and partially purified ␤ne 1:10␮L of protein molecular weight standard;Lane 2:10␮L of crude enzyme;Lane 3:10␮L of enzyme extract.R.N.Barbagallo et al./Enzyme and Microbial Technology41(2007)570–575575than for natural substrate,with a different kinetic behaviour.The enzyme followed Michaelis–Menten kinetics and showed some properties(optimal pH and temperature conditions,glucose competitive inhibition)similar to those found for␤-glucosidase from A.niger[30].Finally,relatively to fruit,it was reported a relation between␤G activity increasing and anthocyanins level decreasing,so it is possible to hypotize the enzyme is involved in anthocyanins degradation reactions duringfinal ripening stage.References[1]Widmer WW,Montanari AM.The potential for citrus phytochemicals inhypernutrition foods.In:Finley JW,Armstrong DJA,Nagy S,Robinson SF, editors.Hypernutritious foods.Auburndale,FL:Agscience,Inc.;1996.p.75–89.[2]Felgines C,Talav´e ra S,Texier O,Besson C,Fogliano V,Lamaison J-L,etal.Absorption and metabolism of red orange juice anthocyanins in rats.Br J Nutr2006;5:898–904.[3]Rapisarda P,Bellomo SE,Intrigliolo F.Anthocyanins in blood oranges:composition and biological activity.Recent Res Dev Agric Food Chem 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[11]Cameron RG,Manthey JA,Baker RA,Grohmann K.Purification and char-acterization of a beta-glucosidase from Citrus sinensis var.Valencia fruit tissue.J Agric Food Chem2001;49:4457–62.[12]AOAC.Official methods of analysis.14th ed.Virginia,USA:Associationof Official Analytical Chemists;1984.p.414–22.[13]Rapisarda P,Intelisano S.Sample preparation for vitamin C analysis ofpigmented orange juices.Ital J Food Sci1996;8:251–6.[14]Rapisarda P,Fallico B,Izzo R,Maccarone E.A simple and reliablemethod for determining anthocyanins in blood orange juices.Agrochimica 1994;38:157–64.[15]Rapisarda P,Fanella F,Maccarone E.Riability of analytical methods fordetermining anthocyanins in blood orange juices.J Agric Food Chem 2000;48:2249–52.[16]Gunata Z,Blondeel,Vallier MJ,Lepoutre,Sapis JC,Watanabe.An endoglycosidase from grape berry skin of cv M.Alexandria, hydrolysing potentially aromatic disaccaride glycosides.J Agric Food Chem1998;46:2748–53.[17]Spagna G,Barbagallo RN,Martino A,Pifferi PG.A method of purify␣-l-arabinofuranosidase and␤-d-glucopyranosidase from Aspergillus niger to increase the aroma wine.Ital Food Bev Technol2000;20:11–6. 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生物质炭（biochar ）属于黑炭的一种类型，是由植物生物质在完全或部分缺氧情况下经热解炭化产生的一类高度芳香化的难熔性固态物质[1]，具有高度热稳定性和较强吸附特性。

据估测，全球每年通过生物体燃烧形成的生物质炭数量在50×1012～200×1012g ，且随着人类活动还将不断上升。

常见的生物质炭包括木炭、秸秆炭、竹炭、稻壳炭等[2]，有研究报道生物质炭主要包括碳（一般高达60%以上）、氢、氧等，其次是灰分（包括钾、钙、钠、镁、硅等）。

据Demirbas 报道，生物质炭的元素组成为：C （66.6%~87.9%）、H （1.2%~2.9%）、O （10.6%~26.6%）[3]。

其元素组成由最终炭化温度决定，随着最终炭化温度的升高，其含碳量增加，氢和氧的含量降低，灰分含量亦有所增加[4]。

Abdul 等利用热农业环境科学学报2011,30(10):2075-2080Journal of Agro-Environment Science摘要：生物质炭是具有高度热稳定性和较强吸附特性的含碳物质，不同来源生物质炭的结构性质可能存在着很大的差异。

为此对2种自制的不同来源生物质炭和1种商业黑炭进行了结构表征，并研究了添加生物质炭对土壤有效养分和腐殖质组成的影响。

结果表明，不同来源的生物质炭在结构上有明显区别：秸秆生物质炭的芳构化程度和热稳定性最低，脂族性最强；商业黑炭缩合程度和热稳定性最高，脂族性最弱；松枝生物质炭介于二者之间。

向土壤中添加秸秆生物质炭和松枝生物质炭培养45d 后，土壤有机碳含量、胡敏酸和富里酸含量、有效养分含量都有不同程度的增加，同时胡敏酸的色调系数Δlg K 降低，对土壤有机碳的长期保存有积极意义。

关键词：生物质炭；结构性质；土壤有效养分；腐殖质组成中图分类号：S153.6文献标志码：A 文章编号：1672-2043(2011)10-2075-06生物质炭结构性质及其对土壤有效养分和腐殖质组成的影响周桂玉，窦森*，刘世杰（吉林农业大学资源与环境学院，长春130118）The Structural Characteristics of Biochar and Its Effects on Soil Available Nutrients and Humus CompositionZHOU Gui-yu,DOU Sen *,LIU Shi-jie（College of Resources and Environmental Science,Jilin Agriculture University,Changchun 130118,China ）Abstract ：Biomass-derived charcoal,also named biochar,has the characteristics of high stability against decay and high capability of adsorp －tion,so it can influence the environment through interaction with climate and geology.There is significant difference in structural characteris －tics of biochar which is obtained from wood and agricultural residues by means of different procedures.As one possible source of the compo －nents with high aromatic structure in soil humus,biochar is of great importance in improving soil fertility,and in maintaining the balance of soil ecosystem.The aim of the research is to investigate the structural characteristics of biochar and the effects of it when applied to soil on available nutrients and humus composition.The results showed that there was great difference between chemical structures of biochar which was produced by different biomass.Aliphatic property of maize straw-derived biochar was stronger than that of pine-derived biochar and commercial black carbon;Aromaticity and thermal stability was on the contrary;Properties of pine-derived biochar was just intermediate.Biochar could influence the soil properties,after being added to soil and incubated for 45days,and the content of soil organic matter,humic acid and fulvic acid were more or less increased to different levels.However,humification degree decreased with the increase of incubated time.The application of biochar could also increase the content of available nutrients,especially the content of phosphorus,alcium and mag －nesium.The decreased Δlg K of humic acid had a positive impact on the long-term preservation of soil organic carbon.Keywords ：biochar;structural characteristics;soil available nutrients;humus composition收稿日期：2011－03－22基金项目：973项目（2011CB100503）；国家自然基金（40871107，40971141）；中国科学院知识创新项目（KZCX2－YW－Q1－07）作者简介：周桂玉（1983—），女，吉林长春人，硕士研究生，从事土壤生物化学的研究。

Genetic Characteristics of the Human Hepatic Stellate Cell Line LX-2Ralf Weiskirchen1*.,Jo¨rg Weimer2.,Steffen K.Meurer1,Anja Kron3,Barbara Seipel3,Inga Vater4, Norbert Arnold2,Reiner Siebert4,Lieming Xu5,Scott L.Friedman6,Carsten Bergmann3,7,81Institute of Clinical Chemistry and Pathobiochemistry,RWTH Aachen University,Aachen,Germany,2Department of Gynaecology and Obstetrics,UKSH Campus Kiel, Kiel,Germany,3Center for Human Genetics,Bioscientia,Ingelheim,Germany,4Institute of Human Genetics,University Hospital Schleswig-Holstein&Christian-Albrechts University Kiel,Kiel,Germany,5Institute of Liver Diseases,Shanghai University of Traditional Chinese Medicine Shuguang Hospital,Shanghai,PR China,6Division of Liver Diseases,Mount Sinai School of Medicine,New York,New York,United States of America,7Department of Human Genetics,RWTH Aachen University,Aachen,Germany, 8Center for Clinical Research,University Hospital Freiburg,Freiburg,GermanyAbstractThe human hepatic cell line LX-2has been described as tool to study mechanisms of hepatic fibrogenesis and the testing of antifibrotic compounds.It was originally generated by immortalisation with the Simian Vacuolating Virus40(SV40) transforming(T)antigen and subsequent propagation in low serum conditions.Although this immortalized line is used in an increasing number of studies,detailed genetic characterisation has been lacking.We here have performed genetic characterisation of the LX-2cell line and established a single-locus short tandem repeat(STR)profile for the cell line and characterized the LX-2karyotype by several cytogenetic and molecular cytogenetic techniques.Spectral karyotyping(SKY) revealed a complex karyotype with a set of aberrations consistently present in the metaphases analyses which might serve as cytogenetic markers.In addition,various subclonal and single cell aberrations were detected.Our study provides criteria for genetic authentication of LX-2and offers insights into the genotype changes which might underlie part of its phenotypic features.Citation:Weiskirchen R,Weimer J,Meurer SK,Kron A,Seipel B,et al.(2013)Genetic Characteristics of the Human Hepatic Stellate Cell Line LX-2.PLoS ONE8(10): e75692.doi:10.1371/journal.pone.0075692Editor:Golo Ahlenstiel,University of Sydney,AustraliaReceived May9,2013;Accepted August20,2013;Published October8,2013Copyright:ß2013Weiskirchen et al.This is an open-access article distributed under the terms of the Creative Commons Attribution License,which permits unrestricted use,distribution,and reproduction in any medium,provided the original author and source are credited.Funding:This work was supported by the German Research Foundation(DFG,SFB/TRR57)and National Institutes of Health(DK56621to SLF).The funders had no role in study design,data collection and analysis,decision to publish,or preparation of the manuscript.Competing Interests:It is correct that three of the listed authors(Carsten Bergmann,Barbara Seipel,and Anja Kron)are employed by a commercial company (Bioscientia Ingelheim,Ingelheim,Germany).The relationship to this company has however not altered the authors’adherence to all the PLOS ONE policies in sharing data and materials.*E-mail:rweiskirchen@ukaachen.de.These authors contributed equally to this work.IntroductionDuring liver fibrogenesis and the establishment of cirrhosis, contractile myofibroblasts(MFB)that originate from quiescent hepatic stellate cells(HSC)are the major source of extracellular matrix(ECM)[1].HSC/MFB possesses the capacity not only for matrix synthesis,but also for the expression and secretion of pro-and anti-inflammatory cytokines and growth factors[2].Based on their pivotal role in the initiation and progression of liver fibrogenesis,HSC/MFB biology is a major focus of fibrosis investigation.However,the preparation of primary cells from the liver is time-consuming and requires special expertise.To overcome these limitations,several spontaneous or experimental-ly-derived immortalized HSC cell lines from mouse,rat and humans have been established[3].Like other permanent cell lines, immortalized stellate cell lines have the advantage of growing continuously to provide unlimited access.Moreover,their clonal origin usually guarantees a considerably homogeneous phenotype that should allow the performance of reproducible experiments in different laboratories[3].Based on this approach,key aspects of stellate cell biology have been uncovered including advances in retinoid metabolism,extracellular matrix expression and turnover,particular,these cell lines are exploited to develop therapeutic approaches.In this context,there is an increasing need for properly characterized stellate cell lines that preserve phenotypic characteristics of HSC/MFB especially for the human derivatives. The human HSC cell lines Lieming Xu-1(LX-1)and Lieming Xu-2(LX-2)were originally generated by transformation of cultured primary HSC obtained from a male human liver with a plasmid encoding the SV40large T-antigen expressed under the control of a Rous sarcoma virus promoter(LX-1)or by spontaneous immortalization of a subset of early passaged LX-1 cells that were grown in low serum conditions(LX-2)[4].Both cell lines express a-SMA,vimentin,the intermediate filament protein glial fibrillary acidic protein(GFAP),and the type b receptor for platelet-derived growth(PDGFR b)suggesting that both cell lines retain key features of activated/transdifferentiated HSC.Both LX cell lines also secrete pro-collagen,pro-MMP-2,MT1-MMP (MMP-14),TIMP-1and TIMP-2,all features characteristic for activated HSC[5].Based on these properties,both LX cell lines have been widely employed as experimental tools in many laboratories worldwide.In particular,LX-2enjoys great popular-ity among researchers interested in the elucidation of mechanismsby the increasing number of publications in which this cell line was utilized.Since the first report in2003[6]and the more detailed characterisation two years later[4],LX-2cells have been cited nowadays in158peer reviewed publications(Figure S1)not only in the field of gastroenterology and hepatology[7–9]but also in studies focused on cellular and molecular biology[10],pharma-cology/toxicology[11],lipid metabolism[12],tissue engineering [13],oncology[14],endocrinology[15]and other general topics [16].Although widely used,LX-2,like all other immortalized cell lines,might be prone to genotypic/karyotypic and phenotypic drifts due to repeated passaging,which may result in cellular sub-lines that are phenotypically and genetically heterogeneous in character.As a consequence,key results obtained with these or other cell lines should be validated in primary cells if possible.Alternatively, further efforts should be made to understand the diverse and heterogeneous outcomes observed with cell lines by more sophisticated characterization of the respective line.With this in mind,we have characterized the genetic profile of LX-2using a number of cytogenetic and molecular methods,including single-locus short tandem repeat(STR)genotyping,standard karyotyp-ing,spectral karyotyping(SKY),and single nucleotide polymor-phism(SNP)array analytics.Our findings indicate that LX-2cells comprise a complex karyotype with various structural abnormal-ities.However,the invariant STR signature and defined specific translocations could be established for LX-2cells.Our results will facilitate proper identity testing,could lead to further mechanistic insights and improve experimental data in the field of liver fibrosis using this cell line.The work also provides importance guidance for the standardized,rigorous evaluation of other cell lines used in experimental hepatology.Materials and MethodsCell CultureHuman hepatic stellate cell line LX-2[4],Wi-38(clone VA-13, subline2RA)(#CCL-75.1,ATCC,Manassas,VA,USA)[17], HepG2[18],and Hep3B[18]were cultured in HEPES-buffered Dulbecco’s modified Eagle medium(DMEM)(Lonza BioWhit-taker,Verviers,Belgium)supplemented with4mmol/L L-glutamine(Lonza),100IU/ml Penicillin/100m g/ml Streptomy-cin(Lonza),and10%(v/v)(Wi-38,HepG2)or2%(v/v)(LX-2) fetal calf serum(FCS)(PAA Laboratories,Pasching,Austria). Human umbilical vein endothelial cells(HUVEC)were isolated from human umbilical cords by collagenase dissociation and grown medium199(Gibco,Life Technologies,Darmstadt, Germany)with20%heat-inactivated fetal bovine serum(FBS) (Gibco),100m g/ml endothelial cell growth supplement(Sigma, Taufkirchen,Germany),and50U/ml heparin(Sigma).Human MFB(hMFB)were isolated as outgrowth from human liver tissues and sub-cultured for several passages as described before[19].All experiments done with LX-2cells were done with passages number between p5and p7in which these numbers refer to those after selection of an immortalized cell population[4]. ImmunocytochemistryApproximately36105LX-2cells were plated on coverslips mounted in6-well dishes and incubated for24h at37u C. Thereafter,the cells were fixed for15min in4%(w/v) paraformaldehyde buffered in phosphate-buffered saline(PBS) (pH7.4)and permeabilized(2min on ice)in0.1%(w/v)sodium citrate/0.1%(v/v)Triton X-100.Then the cells were incubated mapping near the N-terminus of SV40T Ag of SV40origin(sc-20800,Santa Cruz Biotechnology,Santa Cruz,CA),a monoclonal antibody raised against a-smooth muscle actin(a-SMA)or respective normal rabbit(sc-2027,Santa Cruz)or mouse(sc-2343)control IgGs(obtained from Santa Cruz).The antibodies were applied for1h at37u C in1%(w/v)bovine serum albumin in PBS followed by incubation with a goat anti-rabbit IgG-FITC conjugate(sc-2012,Santa Cruz).Cells were mounted in antifade reagent and analysed in UV light using a standard objective. Filamentous actin of LX-2cells was stained with the Alexa Fluor H 488phalloidin conjugate(A12379,Molecular Probes,Invitrogen, Darmstadt,Germany).Nuclei were counterstained with49,6-diamidino-2-phenylindole(DAPI).Western Blot AnalysisProtein extracts were prepared from primary hMFB,LX-2, HepG2,Hep3B,Wi-38,HepG2and HUVEC cells following standard protocols.Equal amounts of proteins were heated at 80u C for10min and separated in4–12%Bis-Tris gels(Invitrogen) under reducing conditions and electro-blotted on nitrocellulose membranes(Schleicher&Schuell,Dassel,Germany).Equal protein loading was monitored in Ponceau S stain and unspecific binding sites were blocked in TBST[10mM Tris/HCl,150mM NaCl,0.1%(v/v)Tween20,(pH7.6)]containing5%(w/v)non-fat milk powder.The membranes were subsequently probed with the antibodies given in Table S1.Primary antibodies were detected with horseradish-peroxidase(HRP)-conjugated secondary antibodies(Santa Cruz)and the Supersignal TM chemiluminescent substrate(Perbio Science,Bonn,Germany).G-banding,SKY and STR AnalysisLX-2cells were trypsinized using Trypsin/EDTA and trans-ferred in multiple T25flasks and cultured for four days.The flask cultures were harvested following standard protocols including treatment with colcemid(10m g/mL),hypotonic KCl solution (0.4%)and fixative(methanol:acetic acid=3:1).G-banding by Pancreatin staining with Giemsa(GPG)was performed following standard procedures.For conventional chromosome analysis,five cells were analyzed by light microscopy and karyotyped using the CytoVision Software(Leica Microsystems).With this method an average banding resolution of250bands was achieved.Aberra-tions were described according to the International Standard of Cytogenetic Nomenclature(ISCN2013)[20].For SKY analysis, metaphases were hybridized with the24-color SKY Paint kit(ASI Applied Spectral Imaging Ltd.,Migdal HaEmek,Israel)according essentially to the manufacturer’s protocol.Spectral images of hybridized metaphases were acquired using the SkyVision II Cytogenetic workstation and analyzed with the SkyView software version1.6(ASI).In addition,images of the DAPI counter-stain of the respective metaphases were acquired as regular CCD images using the direct mode of the SkyVision II system.Inversed and band-enhanced DAPI images were used for the intrachromosomal localization of aberrant chromosome areas.Data from the SKY analysis of LX-2cells was submitted to the public SKY/M-Fish& CGH database platform of the National Cancer Institute(http:// /sky/)that was established for investigators to share and compare their molecular cytogenetic data.For STR multiplex analysis of LX-2cells,two different multiplex assays were taken.The AmpFlSTR Identifiler H PCR amplification kit that amplifies15tetranucleotide repeat loci and the Amelogenin gender-determining marker was obtained from Life Technologies.The PowerPlex H1.2System that detects nine loci(eight STR loci and Amelogenin)was obtained from PromegaSTR profile for LX-2cells was done using the online STR analysis provided by the DSMZ(http://www.dsmz.de)and the ATCC STR Profile database for human cell lines()that actually(April5,2013)contains2455(DSMZ)or1521(ATCC) human PowerPlex H1.2.system-genotyped cell lines.The STR profile of LX-2cells was submitted and deposited in the DSMZ depository.Microarray AnalysisThe Genome-Wide Human SNP Array6.0(Affymetrix,Santa Clara,CA)has been used according to the protocol provided by the manufacturer().Microarrays were washed and stained with the Fluidics Station450(Affymetrix)and scanned with the GeneChip Scanner3000(Affymetrix)using the Genotyping Console software version 4.1(Affymetrix).The Birdseed v2algorithm was used for genotyping.Copy number analysis,loss of heterozygosity(LOH)analysis and segmentation were calculated using Genotyping Console software version4.1. Segments with aberrant copy number were considered as copy number aberration only if they consisted of at least20consecutive SNPs and comprised a minimal size of100kb.Results and DiscussionLX-2Cells Express the SV40Large T-antigen and Markers Characteristic for Activated/transdifferentiated HSCLX-2cells were first introduced in2003[6]and initially characterized in2005[4]as a low-passaged human cell line derived from normal human stellate cells that are spontaneously immortalized.Cells were originally selected by their ability to grow under low serum conditions.The first preliminary description revealed that LX-2cells exhibit typical markers of stellate cells, which in primary culture express desmin and GFAP.Moreover, LX-2cells are responsive to platelet-derived growth factor BB and transforming growth factor-b1,which are the most prominent mitogenic(PDGF-BB)or pro-fibrogenic(TGF-b)stimuli towardsprimary stellate cells,respectively.The expression of a-SMA under all culture conditions indicates that LX-2cells have a partially activated phenotype[4].This conclusion is further supported by their fibroblast-like shape when grown to higher density and their somewhat star-shaped phenotype at lower cell numbers(Figure S2),as well as their high proliferative activity in culture(data not shown).In addition,when early passages of LX-2 cells were stained with fluorescently-labeled phalloidin that binds F-actin with high selectivity and affinity,all cells analysed showed a uniform staining pattern with some preferential expression in distinct areas of the cell membrane and around the nucleus(Figure S3).A similar staining pattern was observed when cells were stained with an antibody directed against a-SMA,confirming their mesenchymal and contractile(i.e.,smooth muscle cell)origin (Figure S3).In the same set of experiments,we further stained early passages of LX-2cells for SV40T Ag that was used for immortalization and found that the nuclei of all cells stained positive for this viral oncogene product demonstrating that all cells within the analysed LX-2culture are more or less homogeneous and further indicates that the T Ag is stably integrated into the genome(Figure S3).The expression of the SV40large T-antigen was also confirmed in Western blot analysis using a rabbit polyclonal antibody raised against amino acids4–30mapping near the N-terminus of SV40T Ag.In this analysis,we used extracts from Wi-38cells as a positive control representing a SV40-transformed human diploid cell line derived from embryonic lung tissue of a female Caucasian that tation antigen[21,22].In line with our immunocytochemical analysis,the Western blot revealed that LX-2cells express low levels of SV40T-Ag(Figure1),while the two human hepatoma-derived cell lines HepG2and Hep3B as well as HUVEC cells were negative for this oncogenic gene product.LX-2cells also express fibronectin,collagen type IV,collagen type I,endoglin,TGF-b receptors type II(T b RII)and I(T b RI), vimentin,desmin,a-SMA,connective tissue growth factor (CTGF),Id2,and p21(Figure1),all representing known pro-fibrogenic matricellular components or mediators involved in hepatic fibrogenesis[23–25].These results partially confirm and extend previous findings demonstrating that LX-2have attributes of primary human HSC in culture[4].In addition,the expression of the tissue inhibitor of metalloproteinase-1(TIMP-1)in LX-2 cells,as previously reported[4],was confirmed(not shown). Conventional and Molecular KaryotypingTo characterize LX-2genetically,we first performed chromo-some G-banding analysis using different LX-2cell cultures.This analysis revealed that LX-2cells have several numerical and structural chromosomal aberrations(Figure2).The karyotype as assessed by this methodology was described as:55,78,der(-X)add(X)(p22.?1),Y[4],del(1)(q4?1)[2],+del(1)(q4?1)[3], der(2;10)(p10;p10)[4],+der(2;10)(p10;p10)[2],del(3)(q25)[3], +del(3)(q25)x2[2],+der(3)add(3)(q2?7)[3], +der(3)?t(3;6)(p25;p2?3)[2],der(4)add(4)(q31)[4],+de-Figure1.Expression analysis.Cell extracts were prepared from hMFB,LX-2,HepG2,Hep3B,HUVEC and Wi-38VA-13subline2RA and analysed in Western blot for expression of(i)extracellular matrix proteins(fibronectin,collagen IV,collagen I),(ii)receptors of TGF-b (endoglin,T b RII,T b RI),(iii)HSC/MFB markers(vimentin,desmin,a-SMA, CTGF,Id2,p21),and(iv)SV40large T-antigen and b-actin.We used two parenchymatic(HepG2,Hep3B)and other cell entities(hMFB,HUVEC, and WI-38)as controls because they are well characterized for the expression of tested genes and served as positive or negative controls in this analysis.doi:10.1371/journal.pone.0075692.g001der(5)?t(5;9)(q35;q34)?del(5)(p15)[5],der(5)add(5)(p1?3)[4],+de-r(5)add(5)(p1?3)x2[2],+6[3],del(7)(p1?3)[3],+del(7)(p1?3)[2],der(9)(11q23-.11q21::14q32-.14q11.2::9p13-.9qter)[2],+der(9)(11q23-.11q21::14q32-.14q11.2::9p13-.9qter)[2],i(10)(q10)[3],del(11)(p11.2)[3],+del(12)(pter-.q12::q15-.q22::q24.2-.qter)[3],213[4],214[4],+15[2],+15[2],der(16)t(16;17)(q11.1;q11.2)[2],+der(16)t(16;17)(q11.1;q11.2)[2],+der(18)(18pter-.18q11.2::1q25-.1q32::7q22-.7qter)[2],+19[2],+20[4],+20[4],+20[2],+21[3],+8,13mar [5][cp5],respectively.In order to further characterize this complex pattern of alterations in more detail,we next performed spectral karyotyping (SKY)that is much more versatile and demanding than conventional chromosome analysis.We detected more than 160chromosomal rearrangements in eight different chromatograms by SKY indicating great genomic heterogeneity of this cell line (Table S2).However,several rearrangements between nonhomologous chromosomes were identified (Figure 3).Notably,translocation products der(9)t(9;11;14),and der(5)t(5;8;14)were frequently observed and especially der(9)t(9;11;14)was present in almost every analysed metaphase (Figure 4,Table S2).In the final SKYGRAM that displays the SKY data of all eight metaphases in the form of coloured chromosome ideograms,it becomes more clear that these chromosomal alterations mainly affect the loss of arm segments in 1q,2p,7p,10q and 14q or rearrangement of individual chromosomes,i.e.der(9)t(9;11;14)and der(8)t(5;8;11),respectively (Figure 5).Based on their common appearance,it is most likely that these rearrangements have already occurred during onset and/or early propagation of this cell line and that they are relatively stable because we observed them in different cell passages (not shown).Subsequently,we performed whole-genome SNP array analysis to confirm the different chromosomal imbalances observed by SKY.This analysis is a useful tool for studying slight variations between whole genomes and provides a signature that is characteristic for each individual.Overall,the generated copy number profile substantially agrees with the described SKYdataFigure 2.Karyogram of LX-2cell.Representative numbers and appearance of chromosomes that was found in one LX-2cell as obtained after Giemsa stain in lightmicroscopic analysis.The karyotype was determined to 74,XYY,+del(1)(q4?1),der(2;10)(p10;p10),+del(3)(q25)x2,+der(4)add(4)(q31),der(5)?t(5;9)(q35;q34)?del(5)(p15),der(5)add(5)(p1?3),+der(5)add(5)(p1?3)x2,+6,del(7)(p1?3),+del(7)(p1?3),+der(9)(11q23-.11q21::14q32-.14q11.2::9p13-.9qter)x3,+i(10)(q10),211,del(11)(p11.2),+del(12)(pter-.q12::q15-.q22::q24.2-.qter),213,214,+15,+der(16)t(16;17)(q11.1;q11.2),+der(18)(18pter-.18q11.2::1q25-.1q32::7q22-.7qter),+20,+20,+21,+21,der(22)?t(22;?)(q11.2;?),+11mar,respec-tively.Please note that a characteristic derivative of chromosome 18,i.e.+der(18)(18pter-.18q11.2::1q25-.1q32::7q22-.7qter),is marked with an arrow in this figure.A characteristic derivative of chromosome 18that contains part of chromosomes 1and 7was seen in three karyograms and is marked by a black arrow.Figure3.Spectral karyogram(SKY)of the hepatic stellate cell line LX-2.Shown is a representative image of LX-2cells hybridized with a24-color SKY probe(upper left panel).The chromosomes were counterstained with DAPI(upper right panel).The spectral images of hybridized metaphases were acquired using a cytogenetic workstation and grouped with the SkyView software(lower panel).Based on this analysis,the karyotype of this LX-2metaphase is determined to:73,3n+.,XXXYY,ish+der(1)t(1;18)(p11;q21)t(1;7)(q42;p12),der(2)t(2;10)(p11;q11),del(2)(q11), +der(3)t(3;6)(qter;q11),der(3)t(3;5)(p11;q23)t(3;5)(q24;q23)del(5)(q14),25,+del(6)(p11),der(6)t(6;14)(p11;q11),der(6)t(6;11)(q11;q12), der(7)t(7;18)(p11;p11),del(7)(q21),+der(8)t(8;5)(q11;q11)t(5;14)(q31;q23),der(8)t(4;8)(q32;p12),+der(9)t(3;9)(p22;qter)del(9)(p11), der(9)t(9;14)(p12;q11)t(14;11)(q24;q14)del(11)(q12),+der(10)del(10)(p12)del(10)(q23),der(10)t(5;10)(p12;p11),del(11)(p14),der(11)del(11)(p14)-del(11)(q23)x2,der(12)t(12;22)(q13;q12),213,214x2,+i(15)(p10),+der(15)t(15;20)(q15;q11),der(16)t(16;17)(p11;q11),del(17)(p11),219,220,221, der(21)t(21;13)(p11;q11),del(X)(q24),del(X)(q25),del(X)(q21).Please note that the derivative that contains parts of chromosomes1,18and7(cf. Figure1)were also seen in this analysis.doi:10.1371/journal.pone.0075692.g003Figure4.Median appearance of chromosome regions per cell based upon SKY analysis of eight metaphases from human LX-2cell line.The average copy numbers per cell of each human chromosome region are shown in correspondence to their chromosome ideogram based onsummary SKY analysis.(cf.Figure4and6).The comparison of results obtained in SKY (Figure4)and SNP array analysis(Figure6)indicates similar chromosomal gains in3q,5p,5q,11q,14q,17q,20and losses in 1q,7p,8q,9p,and11p.Therefore,the SKY results of the eight metaphases seem to reflect a representative intersection of the complete culture genome used for microarray analysis.It is important to note that the LX-2cell line was originally established from primary HSC that were transfected with the large SV40T-antigen in passage4[4].Subsequently,transfected cells were further passaged seven times before an individual clone(LX-1)was finally established.Afterwards LX-2was expanded as a cellular subline that was able to grow under reduced serum selection conditions induced several independent subclonal rear-rangements that were simultaneously passed during prolonged culturing and passages resulting in cellular heterogeneity.We presently do not known if the observed translocations or imbalances might be of relevance for the fibrogenic activities of LX-2or if they are simply induced by the immortalization using the large T-antigen.In addition,SKY in our analysis does not allow concise molecular definition of the respective breakpoints to the gene level,so that we do not know if these rearrangements introduce alterations in gene expression or are causative for the establishment of the immortalized phenotype of this cell line. STR analysis.Inter-and intraspecies cross-contamination inplete SKYGRAM of LX-2cells based on eight cells.Characters above rearranged chromosome ideograms are for numbers of metaphases with this aberration.Rearrangements recognized only once in SKY analysis are ignored.Based on this analysis,the complete SKYGRAM of LX-2was determined to64,83,n3+/2.,XXY,21,del(1)(q11)[3],del(2)(q11)[3],+del(3)(p11)[2],del(6)(p11)[2],del(7)(p11)[4],del(7)(q11)[2], del(7)(p15)[2],+der(7)t(7;18)(p11;p11)[3],der(8)t(5;8)(q11;q11)t(5;14)(q31;q23)[4],der(9)t(9;14)(p12;q11)t(11;14)(q14;q24)del(11)(q12)[7],del(10)(q22) [4],i(10)(q10)[2],der(11)del(11)(p11)del(11)(q22)x2[2],del(12)(q11)[2],+del(13)(q21)[2],-14,-14,+del(17)(p11)[2],+20[cp8],respectively.More detailed SKYGRAM data for LX-2cells were deposited in the SKY/M-FISH&CGH database located at the National Center for Biotechnology Information that can be found at:/sky/skyquery.cgi.doi:10.1371/journal.pone.0075692.g005and a frequent cause of false experimental outcome and scientific misinterpretation.Although this is already recognized for over60 years[26–31],urgent appeals to scientists to address this issue have not been widely accepted,including studies of the liver[32]. However,most cell lines(i.e.HeLa)that have already contributed for over60,000publications are still incompletely characterized [33].Therefore,it was recently proposed that the unambiguous cell authentication in studies using cell lines should be a consideration during peer review of papers submitted for publication or during review of grants submitted for funding[34]. To address this problem,an international expert team of scientists has therefore prepared a consensus standard for the authentication of human cells using STR profiling strategies[35]. This approach provides a simple method for rapid and cheap cell line identification[35]and further enables the establishment of a public STR profile database under the auspices of the National Center for Biotechnology information[30].Consistent with these recommendations,we next performed STR profiling for LX-2cells and all other cell lines used in this study by using two different commercially available kit systems,As expected,both kits revealed a male genotype in LX-2(Table S3). In addition,LX-2were genotyped to be homozygous for the STR loci D16S539(13),D7S820(11),TH01(9.3),vWA(17),D8S1179 (13),D2S1338(17),D18S51(12)and to be heterozygous for markers D13S317(11,13),D5S818(11,12),CSF1PO(10,12), TPOX(8,9),D21S11(28,31),D3S1358(13,15),D19S433(13, 15.2),and FGA(21,26),respectively(Table S3).A detailed STR provided by the DSMZ or ATCC revealed that this STR profile is unique for LX-2and not deposited yet in these data bases.The STR profiles for HepG2,Hep3B and Wi-38were identical to those that are given in respective ATCC and DSMZ depositories (for details see Material and Methods).Expression analysis further revealed that LX-2cells express typical markers of HSC/MFB including fibronectin,collagen type IV,collagen type I,various TGF-b receptors(i.e.endoglin,T b RII, T b RI),vimentin,desmin,a-smooth muscle actin,CTGF,and p21 suggesting that this cell line is indeed originated from hepatic stellate cells(Figure1).It is presently not possible to predict the impact of the observed genetic alterations for studies performed in LX-2cells.However, the observed chromosomal aberrations and the definition of a unique,characteristic STR profile for LX-2will now help to fulfil the recommendation for cellular authentication required by granting agencies and scientific journals when working with LX-2and to avoid unreliable experimental outcomes and scientific misinterpretation.Hopefully,our study increase the value and suitability of LX-2cells in liver research and provides an important benchmark for proper genetic characterization of widely used cell lines in the field of hepatology.Supporting InformationFigure S1Usage of LX-2cells during2003–2013.StudiesFigure6.Chromosomal imbalances detected in the LX-2cell line using a SNP6.0Array.Gains and losses are shown by red and blue triangles,respectively.doi:10.1371/journal.pone.0075692.g006。

2612 ｜中国组织工程研究｜第25卷｜第16期｜2021年6月生物可吸收材料特点及在骨科中的应用李晏乐1，岳肖华1，聂 真2，张峻玮1，李朝辉1，聂伟志1，姜红江1文题释义：生物可吸收材料：是指能够在体内生物环境中被降解和吸收的材料，因其用于制作人体植入装置可以避免植入物长期植入人体组织导致炎症或其他症状的发生及减轻患者二次手术的痛苦，使其在生物医学多个领域中得到广泛应用。

生物相容性：指生命体组织对非活性材料产生反应的一种性能，一般是指材料与宿主之间的相容性。

生物材料植入人体后对特定的生物组织环境产生影响和作用，生物组织对生物材料也会产生影响和作用，两者的循环作用一直持续，直到达到平衡或者植入物被去除。

摘要背景：生物可吸收材料在十几年的临床应用中展现了明显优势，在生物医学多个领域中得到广泛应用。

目的：综述现阶段生物可吸收材料的特点及其在骨科中的应用进展。

方法：应用计算机检索万方数据库、CNKI 数据库、维普数据库和PubMed 数据库中的相关文献，中文检索词为“生物可吸收材料、生物可吸收金属材料、生物可吸收无机材料、高分子材料、生物复合材料”，英文检索词为“Bioabsorbable/Bioabsorbable material 、Metal material 、Polymer material 、Biocomposites ”。

结果与结论：可吸收金属材料具有较好的机械性能；聚合物材料腐蚀机制明确，可以预测其在体内外腐蚀行为和腐蚀速率，但承重性能不如可吸收金属材料；生物陶瓷材料经过一定处理后具有良好的生物相容性、骨传导性及骨结合性，但脆性大且不易成型；生物复合材料不仅兼具组分材料的性质，还能获得单一组分材料所不具有的新性能，具有广泛的应用的前景。

关键词：骨；材料；生物可吸收；高分子材料；生物复合材料；生物相容性；骨组织工程；综述Characteristics and application of bioabsorbable materials in orthopedicsLi Yanle 1, Yue Xiaohua 1, Nie Zhen 2, Zhang Junwei 1, Li Zhaohui 1, Nie Weizhi 1, Jiang Hongjiang 11Wendeng Orthopedic Hospital of Shandong Province, Weihai 264400, Shandong Province, China; 2School of Materials Science and Engineering, Beijng Institute of Technology, Beijing 100081, ChinaLi Yanle, Master, Physician, Wendeng Orthopedic Hospital of Shandong Province, Weihai 264400, Shandong Province, ChinaCorresponding author: Nie Weizhi, Chief physician, Wendeng Orthopedic Hospital of Shandong Province, Weihai 264400, Shandong Province, ChinaCo-corresponding author: Jiang Hongjiang, Chief physician, Wendeng Orthopedic Hospital of Shandong Province, Weihai 264400, Shandong Province, ChinaAbstractBACKGROUND: Bioabsorbable materials have shown obvious advantages in clinical application for more than ten years, and have been widely used in many biomedical fields.OBJECTIVE: To review characteristics of bioabsorbable materials and their application in orthopedics.https:///10.3969/j.issn.2095-4344.3148投稿日期：2020-05-13送审日期：2020-05-16采用日期：2020-06-17在线日期：2020-10-21中图分类号： R459.9；R318.08；R-1文章编号：2095-4344(2021)16-02612-06文献标识码：A1山东省文登整骨医院，山东省威海市 264400；2北京理工大学材料学院，北京市 100081第一作者：李晏乐，女，1993年生，湖北省荆州市人，汉族，2019年中国中医科学院毕业，硕士，医师，主要从事中西医结合治疗骨与关节疾病方面的研究。

高二英语生物分类单选题50题1. Which of the following belongs to the phylum Arthropoda?A. EarthwormB. StarfishC. ButterflyD. Sponge答案：C。

解析：节肢动物门（Arthropoda）的典型特征包括具有分节的附肢等。

蝴蝶（Butterfly）属于节肢动物门。

蚯蚓（Earthworm）属于环节动物门 Annelida）。

海星 Starfish）属于棘皮动物门Echinodermata）。

海绵 Sponge）属于多孔动物门 Porifera）。

2. The organism which is classified in the class Mammalia should have the following feature:A. Gills for breathingB. Feathers on the bodyC. Hair or fur and produce milk to feed their youngD. Scales on the body答案：C。

解析：哺乳纲（Mammalia）的生物具有毛发或皮毛并且能够产奶哺育幼崽。

用鳃呼吸（Gills for breathing）是鱼类等水生生物的特征，它们属于鱼纲等，不属于哺乳纲。

身上有羽毛（Feathers on the body）是鸟类的特征，鸟类属于鸟纲（Aves）。

身上有鳞片（Scales on the body）是爬行动物等的特征，爬行动物属于爬行纲（Reptilia）。

3. Which kingdom does the mushroom belong to?A. AnimaliaB. PlantaeC. FungiD. Protista答案：C。

解析：蘑菇属于真菌界（Fungi）。

动物界（Animalia）的生物具有能运动、异养等特点。