固定化微球降解土壤中PAEs效果及影响因素

旨为研究土壤邻苯二甲酸酯污染修复中,固定化微球降解土壤中邻苯二甲酸酯的效果及影响因素。以海藻酸钠为载体,采用包埋法对课题组前期提取的微小杆菌进行固定化,比较固定化微球和游离菌降解土壤中邻苯二甲酸酯(Phthalates esters,PAEs)的效果及pH、温度、重金属、无机盐等对降解菌降解目标物的影响。结果显示:(1)在土壤环境相同条件下,固定化微球对邻苯二甲酸二甲酯(Dimethyl ortho-phthalate,DMP)、邻苯二甲酸二正丁酯(Di-n-butyl ortho-phthalate,DnBP)和邻苯二甲酸二(2-乙基己)酯(Bis(2-ethylhexyl)ortho-phthalate,DEHP)的降解效果高于游离菌,DMP在7 d可降解完全,DnBP在10 d内可降解完全,DEHP在20 d降解率63.73%;而游离菌则在15 d内完全降解DMP,20 d内完全降解DnBP,DEHP在20 d降解率48.77%;(2)不同pH值时,固定化微球对DMP、DnBP、DEHP的降解率均高于游离菌,pH9时,固定化微球对于DMP、DnBP、DEHP的降解率最高分别为96.81%、89.39%、58.35%;(3)不同温度,固定化微球对DMP、DnBP、DEHP的降解率也均高于游离菌,温度为30℃时,固定化微球对于DMP、DnBP、DEHP的降解效率达到最高,分别为96.27%、89.19%、59.01%;(4)重金属使游离菌对DMP、DnBP、DEHP降解率下降较多,而使固定化微球对DMP、DnBP的降解率仅下降了16.35%、9.95%,DEHP不仅没有降低,反而增加2.49%,说明重金属对游离菌起到很强的抑制作用,但对于固定化微球的降解效果影响较小;(5)盐碱条件下,中性盐极大降低了游离菌和固定化微球降解DMP、DnBP、DEHP的降解能力,碱性盐和混合盐对降解菌影响较小,且增强了固定化微球对DnBP、DEHP的降解能力。固定化微球降解PAEs效果明显高于游离菌,对外界环境有更好的适应能力,且对重金属、无机盐污染环境有一定的抵御能力。     

聚丁二酸丁二醇酯高效降解菌的筛选及降解特性

从蔬菜地表微环境空气微生物中分离筛选获得了一株高效降解聚丁二酸丁二醇酯(PBS)的真菌菌株BFM-X1, 通过形态学和ITS rDNA 序列测定分析, 确认该菌株为淡色生赤壳属真菌(Bionectria ochroleuca)。该菌株降解PBS 薄膜的最适温度为25 °C?30 °C, 培养基初始pH 为4.0, 以甘油、大豆油、葡萄糖及PBS 乳剂分别作为唯一碳源时对PBS 薄膜均具有很高的降解率; 菌株对PBS 薄膜的降解率随PBS 乳剂含量变化的曲线呈倒“U”型, 最佳PBS 乳剂浓度为1 g/L, 且对PBS 的降解呈现诱导期、指数降解期和加速降解期等3 个阶段。降解初期薄膜表面首先粗糙化, 失去原有的黑色光泽, 随后出现孔洞, 逐渐呈现破碎化, 最后薄膜被完全降解, 降解部位仅残留黑色色素。     

一株阿特拉津降解菌的分离鉴定及降解特性

从农药厂废水处理池的活性污泥中分离到一株阿特拉津降解菌X-4, 根据其生理生化特性和16S rRNA基因序列相似性分析, 将其初步鉴定为节杆菌属(Arthrobacter sp.)。该菌能以阿特拉津为唯一碳氮源生长, 42 h内对100 mg/L的阿特拉津降解效果为95.7%, 降解阿特拉津的最适温度为30 °C, pH为7.0。该菌对多种重金属离子都存在抗性, 显示了其在去除阿特拉津和重金属复合污染方面的应用潜力。对其降解基因的初步研究显示, 该菌含有trzN、atzB和atzC 3个阿特拉津降解相关基因。     

近海柴油降解菌群的构建及其对柴油的降解特性

【目的】实施近海柴油污染的生物治理。【方法】以柴油为唯一碳源,从深圳港口海域富集筛选柴油降解菌;采用复配、正交试验等方式构建混合菌群;通过单因素试验研究环境因素对菌群降解柴油的影响;使用气相色谱-氢火焰检测器(GC-FID)分析降解前后柴油各组分的变化;通过生理生化试验和16S rRNA基因序列分析对菌株进行鉴定。【结果】获得了16株柴油降解菌,7 d内对柴油的降解率最高达40.8%;选择菌株C1-8、C2-10、C3-13构建了混合菌群CQ1,投加量分别为0.5%、2.0%和1.0%,CQ1对柴油去除率比单菌提高了10%以上;CQ1的最适环境条件为：温度30 °C、pH 7.6、摇床转速220 r/min、柴油浓度20 g/L,优化后9 d内对柴油去除率达60%以上;GC-FID结果显示,菌群CQ1可降解大部分C11?C27的正构烷烃,对C21?C27的烷烃降解可达100%。经鉴定,菌株C1-8、C2-10和C3-13分别为微杆菌(Microbacterium sp.)、剑菌(Ensifer sp.)和变异棒杆菌(Corynebacterium variabile)。【结论】CQ1在近海柴油污染的生物修复中具有良好的应用前景。     

一株高效DEHP降解菌的分离、鉴定及其降解特性

【目的】分离得到高效的邻苯二甲酸二乙基己基酯(DEHP)降解菌。【方法】采用富集培养法筛选分离菌株,并对菌株进行驯化;通过PCR扩增得到其16S rRNA和gyrB基因序列,进行同源序列分析及分子系统发育树的构建,同时结合形态学观察和生理生化实验对菌株进行初步鉴定;采用高效液相色谱(HPLC)分析菌株对DEHP的降解特性。【结果】分离得到一株能以DEHP为唯一碳源和能源生长的菌株,命名为HS-NH1,初步鉴定其为戈登氏菌(Gordoniasp.)。菌株HS-NH1最适的生长和降解条件为30°C、pH 7.0,在此条件下,该菌株60 h内能够将浓度为500 mg/L的DEHP降解90%以上。高效液相色谱(HPLC)分析表明,菌株HS-NH1在降解DEHP过程中产生了一种重要的中间代谢产物——邻苯二甲酸。底物广谱性试验证明,菌株HS-NH1能够有效地利用多种常见的邻苯二甲酸酯(PAEs)与芳香族衍生物。【结论】筛选得到了一株DEHP降解菌Gordonia sp.HS-NH1,该菌降解效率高,具有良好的底物广谱性,在邻苯二甲酸酯类化合物的污染治理中将会有一定的应用潜力。     

降解尿酸乳酸菌复合菌系的筛选与菌株组合提升降解效果

【背景】高尿酸症由血液中尿酸含量明显升高而导致,利用乳酸菌对人体的益生作用缓解高尿酸血症越来越受到关注。【目的】获得具有降解尿酸能力的乳酸菌复合菌系与纯培养菌株。【方法】以泡菜为样品来源,以尿酸为底物,采用MRS培养基筛选降解尿酸的乳酸菌复合菌系,通过高效液相色谱法测定复合菌系对尿酸的降解能力。【结果】得到一组乳酸菌复合菌系,当培养温度为37 °C、pH值为6.20、静置培养72 h后复合菌系对尿酸的降解率为12.08%;通过优化培养条件,当该菌系在以牛肉膏为单一氮源、初始pH值为5.00、温度为35 °C的条件下培养72 h,尿酸降解率上升至17.19%,降解率比优化前提高了42.3%;从该菌系中分离出两株具有尿酸降解能力的菌株UA-1与UA-2,它们的尿酸降解率分别为10.85%和8.65%;通过形态学观察和16S rRNA基因序列分析,经鉴定两株菌均为布氏乳杆菌(Lactobacillus buchneri)。将两株单菌组合降解尿酸试验发现,UA-1与UA-2比例为2:1的尿酸降解率为20.2%,比原复合菌系的降解能力提高了67.22%。【结论】研究证明了乳酸菌复合菌系对尿酸的降解能力优于单个菌株,为后续利用乳酸菌复合菌系应用提供了数据支持。     

阿维菌素在土壤中的微生物降解研究

运用恒温培养法研究了阿维菌素在土壤中的降解动力学．结果表明,非生物+微生物降解、非生物降解及微生物降解的半衰期分别为34．8、277．3和49．9d,说明阿维菌素在土壤中的降解主要由微生物引起．从试验土壤中分离到1株高效降解阿维菌素的菌株,经16S rDNA鉴定为嗜麦芽寡养单胞菌(Stenotrophomonas maltrophilia)．从该降解菌中提取的粗酶液米氏常数(Km)为6．78nmol·ml^-1,最大降解速率为81．5nmol·min^-1·mg^-1。     

一株瘤胃纤维素降解菌的分离鉴定及其纤维素降解特性

从蒙古绵羊瘤胃内容物中分离到一株纤维素降解细菌WH-1, 通过形态、生理生化特征、G+C mol%含量和16S rRNA序列分析对分离菌株进行鉴定, 鉴定为溶纤维丁酸弧菌属(Butyrivibrio fibrisolvens)的溶纤维丁酸弧菌(Butyrivibrio fibrisolvens)。同时, 用Mega 4.1软件构建的系统发育树显示分离菌株WH-1与多株溶纤维丁酸弧菌(Butyrivibrio fibrisolvens)的亲缘关系最近。对该菌株纤维素降解特性的初步研究表明：当温度为37°C、     

一株原油降解菌的分离鉴定及降解特性研究

[目的]对从大连湾原油污染海域生长的海绵中分离的原油降解菌2-9进行鉴定及降解特性研究.[方法]采用16S rRNA基因序列同源性分析、生理生化指标测定、DNAG+C含量测定、全细胞脂肪酸组成测定、碳源利用实验等多种方法对该菌株进行鉴定,并通过降解实验测定其对原油的降解情况.[结果]菌株2-9鉴定为Nitratireductor basaltis,革兰氏阴性,接触酶和氧化酶阳性.在GenBank中与其16S rRNA基因序列相似度最高的模式株为Nitratireductor basaltis J3T,相似性为99％.可生长的pH范围为6.0-10.0,最适生长pH值为8.0;可生长温度范围为15℃-42℃,最适生长温度为30℃; NaCl浓度生长范围是0-8％(W/V),最适生长盐度为2％.该菌株可以利用多种糖和有机酸的碳源,其DNA G+C含量为57.29 mol％,主要脂肪酸组成为ω7c-十八碳单不饱和脂肪酸(63.61％)、ω8c型环式十九碳饱和脂肪酸(16.97％)、饱和十八碳脂肪酸(4.28％)和十六碳饱和脂肪酸(3.39％).同时,考察了该菌株对原油的降解效果,在人工海水培养基中,14d内对原油(初始浓度为1 g/L)的平均降解率为63.5％.[结论]菌株2-9是一株具有开发潜力的原油降解菌.     

对硝基苯酚降解菌Pseudomonas sp. PDS-7的降解特性及 其降解相关基因的克隆

[目的]本研究的目的是分离对硝基苯酚(PNP)降解菌,研究其对PNP的降解特性;克隆其降解相关基因,并进行表达.[方法]本研究通过富集培养法和系列稀释平板涂布法分离PNP降解菌株;采用形态观察、生理生化特征测定和16S rDNA分析对菌株进行初步鉴定;通过摇瓶试验研究菌株降解特性;利用SEFA-PCR技术克隆降解相关基因,并亚克隆到表达载体pET29a中,构建重组表达质粒pETpnpC,再转入受体菌E.coli BL21(DE3)中进行诱导表达;通过分光光度法测定表达产物的酶活力.[结果]分离到一株PNP降解菌PDS-7,将该菌株鉴定为假单胞菌属(Pseudomonassp.);该菌株能够以PNP作为唯一碳源、氮源和能源生长,菌株对PNP的最高耐受浓度为80 mg/L,最适降解温度为30℃,偏碱性条件有利于菌株对PNP的降解;克隆了PNP降解过程中的偏苯三酚1,2-双加氧酶基因pnpC及马来酰醋酸还原酶基因pnpD(GenBank登陆号EU233791);将pnpC在E.coli BL21(DE3)菌株进行了诱导表达,表达产物对偏苯三酚和邻苯二酚均有邻位开环活性,比活力分别为0.45 U/mg protein和0.37 U/mg protein,表明偏苯三酚1,2-双加氧酶基因pnpC得到了活性表达.[结论]分离鉴定了一株PNP降解菌Pseudomonas sp.PDS-7,研究了该菌株的降解特性,克隆和表达了降解相关基因.     

Biodegradation of di-n-butyl phthalate by Rhodococcus sp. JDC-11 and molecular detection of 3, 4-phthalate dioxygenase gene

Rhodococcus sp. JDC-11, capable of utilizing di-n-butyl phthalate (DBP) as the sole source of carbon and energy, was isolated from sewage sludge and confirmed mainly based on 16S rRNA gene sequence analysis. The optimum pH, temperature, and agitation rate for DBP degradation by Rhodococcus sp. JDC-11 was 8.0, 30 degrees C, and 175 rpm, respectively. In addition, the effect of glucose concentration on DBP degradation indicated that low concentration of glucose inhibited the degradation of DBP while high concentrations of glucose increased its degradation. Meanwhile, the substrates utilization test showed that JDC-11 could also utilize other phthalates. Furthermore, the major metabolites of DBP degradation were identified as mono-butyl phthalate and phthalic acid by gas chromatography-mass spectrometry and the metabolic pathway of DBP degradation by Rhodococcus sp. JDC-11 was tentatively speculated. Using a set of new degenerate primer, partial sequence of the 3, 4-phthalate dioxygenase gene was obtained from the strain. Sequence analysis revealed that the phthalate dioxygenase gene of JDC-11 was highly homologous to the large subunit of phthalate dioxygenase from Rhodococcus coprophilus strain G9.     

Isolation and characterization of marine diesel oil-degrading Acinetobacter sp. strain Y2

The marine diesel oil-degrading bacterium Acinetobacter sp. strain Y2 was isolated from oil-polluted seawater sampled from Dinghai port, Zhoushan City, Zhejiang Province, China. The isolated bacterium was identified as Acinetobacter sp. based on its 16S rDNA gene sequence as well as various morphological and physiological characteristics. The degradation characteristics of strain Y2 were studied and its parameters for oil degradation optimized. These optimal conditions were determined to be an initial pH of 7.5, an incubation temperature of 30 °C, an initial diesel oil concentration of 2 % (v/v), and an initial inoculating bacteria concentration of 3?×?10⁷ cells/mL. The results from the gas chromatography–mass spectrometry analysis showed that strain Y2 could almost completely degrade all components of diesel oil, with a degradation ratio of up to 80 % after 10 days of incubation at the optimal conditions.     

Degradation of environmental endocrine disruptor di-2-ethylhexyl phthalate by a newly discovered bacterium, Microbacterium sp. strain CQ0110Y

In this study di-2-ethylhexyl phthalate (DEHP)-degradation strain CQ0110Y was isolated from activated sludge. According to the biophysical/biochemical characteristics and analysis of 16S rDNA, the strain was identified as Microbacterium sp. The results of this study showed the optimal pH value and optimal temperature which influenced the degradation rate in wastewater: pH 6.5–7.5, 25–35°C. Kinetics of degradation reaction had been performed at different initial concentrations and different time. Analyzed with SPSS10.0 software, the DEHP degradation can be described as the same exponential model when the initial DEHP concentration was lower than 1,350 mg/l. The kinetics equation was ln C = −0.4087t + A, with the degradation half life of DEHP in wastewater (1.59 days). To the best of our knowledge, this is the first reported case of DEHP degradation by Microbacterium sp. strain. Xiang Li and Ji-an Chen contributed equally to this work.     

Purification and characterization of a thermostable laccase with unique oxidative characteristics from <Emphasis Type="Italic">Trametes hirsuta</Emphasis>

A laccase was purified from Trametes hirsuta. This laccase was classified as a “white” or “yellow” laccase. pH 2.4 was optimal for the oxidation of ABTS and pH 2.5 for DMP. DMP oxidation was optimal at 85°C. The half-life of this laccase was 70 min at 75°C, and 5 h at 65°C. Non-phenolic dyes, such as Methyl Red, were oxidized by purified laccase without mediators. The enzyme was not inhibited by Cu²⁺, Mn²⁺, or EDTA. These are atypical laccase characteristics that make it a good candidate for theoretical and applied research.     

Isolation and identification of Paracoccus sp. FD3 and evaluation of its formaldehyde degradation kinetics

A formaldehyde-degrading bacterium strain, FD3, was isolated from contaminated soil and identified as Paracoccus sp. based on partial 16S rRNA gene sequence analysis. In batch culture, the bacterium metabolized 5,000 and 8,000 mg/L formaldehyde completely within 16 and 18 h, respectively, at 30°C (pH 7.0) with agitation at 150 rpm. The degradation kinetics was found to follow a first-order model at all initial formaldehyde concentrations with regression values greater than 0.99. Formaldehyde degradation rates increased from 532.37 to 2283.04 mg/L/h as the initial concentration of formaldehyde was increased from 1,000 to 8,000 mg/L. The growth of strain FD3 on formaldehyde as a sole carbon and energy source was well described by the Luong model with a maximal specific growth rate of 0.1754/h, a half-saturation constant of 309.02 mg/L, and a maximum substrate concentration of 3875.53 mg/L. Due to its high tolerance and degradation capacity to formaldehyde, Paracoccus sp., FD3 is considered an excellent candidate for use in degrading formaldehyde in wastewaters.     

Degradation of the endocrine-disrupting dimethyl phthalate carboxylic ester by Sphingomonas yanoikuyae DOS01 isolated from the South China Sea and the biochemical pathway

Bacteria capable of using dimethyl phthalate (DMP) as the sole carbon and energy source were isolated from the sediments collected at a depth of 1340 m from the South China Sea. Sphingomonas yanoikuyae DOS01, identified based on 16S rRNA gene sequence, utilized DMP from an initial level of 180 mg l^?1 to non-detectable in 35 h at 30 °C, the optical density (OD₆₀₀) values increased over the time of incubation. Degradation intermediate monomethyl phthalate (MMP) accumulated up to 21.3 mg l^?1 and then disappeared in the culture medium. When MMP or another intermediate phthalate (PA) was used as the sole substrate, this strain was only capable of degrading MMP, but not PA. Total organic carbon (TOC) analysis of the culture medium suggested that both DMP and MMP were mineralized, but not PA. This strain from the deep-ocean sediment transforms DMP to MMP using a common biochemical pathway for DMP as reported before. Further esterase activity assays indicated that the enzyme induced by MMP has higher affinity than that by DMP for the substrate p-nitrophenyl acetate. Our results indicated that complete degradation of DMP by this marine microorganism may involve a new biochemical pathway.     

Degradation of cationic surfactants using Pseudomonas putida A ATCC 12633 immobilized in calcium alginate beads

In this study, the degradation of tetradecyltrimethylammonium bromide (TTAB) by freely suspended and alginate-entrapped cells from the bacteria Pseudomonas putida (P. putida) A ATCC 12633 was investigated in batch cultures. The optimal conditions to prepare beads for achieving a higher TTAB degradation rate were investigated by changing the concentration of sodium alginate, pH, temperature, agitation rate and initial concentration of TTAB. The results show that the optimal embedding conditions of calcium alginate beads are 4 % w/v of sodium alginate content and 2 × 10⁸ cfu ml^?1 of P. putida A ATCC 12633 cells that had been previously grown in rich medium. The optimal degradation process was carried out in pH 7.4 buffered medium at 30 °C on a rotary shaker at 100 rpm. After 48 h of incubation, the free cells degraded 26 mg l^?1 of TTAB from an initial concentration of 50 mg l^?1 TTAB. When the initial TTAB concentration was increased to 100 mg l^?1, the free cells lost their degrading activity and were no longer viable. In contrast, when the cells were immobilized on alginate, they degraded 75 % of the TTAB after 24 h of incubation from an initial concentration of 330 mg l^?1 of TTAB. The immobilized cells can be stored at 4 °C for 25 days without loss of viability and can be reused without losing degrading capacity for three cycles.     

Production of extracellular alkaline lipase by a new thermophilicBacillus sp

A thermophilic soil isolate—Bacillus sp. RS-12, grew optimally at 50°C and not below 40°C. Production of an extracellular lipase by this organism was substantially enhanced when the type and concentration of carbon and nitrogen sources and initial pH of the culture medium were consecutively optimized. The lipase production was found to be growth-associated with maximum secretion in the late exponential growth phase,i.e. 15h of incubation. The enzyme activity as high as 0.98 nkat/mL was obtained under optimum conditions. Tween 80 (0.5%) and yeast extract (0.5%) were found to be the best carbon and nitrogen sources inducing maximum enzyme yield with initial pH 8.0 at 50°C. The kinetic characteristics of the crude lipase indicated the highest activity at 50–55°C and pH 8.0. It had a half life of 60, 18 and 15 min at 65, 70 and 75°C, respectively.     

Immobilization of Bacillus licheniformis L-Arabinose Isomerase for Semi-Continuous L-Ribulose Production

Bacillus licheniformis L-arabinose isomerase (BLAI) with a broad pH range, high substrate specificity, and high catalytic efficiency for L-arabinose was immobilized on various supports. Eupergit C, activated-carboxymethylcellulose, CNBr-activated agarose, chitosan, and alginate were tested as supports, and Eupergit C was selected as the most effective. After determination of the optimum enzyme concentration, the effects of pH and temperature were investigated using a response surface methodology. The immobilized BLAI enzyme retained 86.4% of the activity of the free enzyme. The optimal pH for the immobilized BLAI was 8.0, and immobilization improved the optimal temperature from 50 °C (free enzyme) to a range between 55 and 65 °C. The half life improved from 2 at 50 °C to 212 h at 55 °C following immobilization. The immobilized BLAI was used for semi-continuous production of L-ribulose. After 8 batch cycles, 95.1% of the BLAI activity was retained. This simple immobilization procedure and the high stability of the final immobilized BLAI on Eupergit C provide a promising solution for large-scale production of L-ribulose from an inexpensive L-arabinose precursor.     

一株低温玉米秸秆降解真菌的筛选、鉴定及降解特性

【背景】在我国北方地区玉米秸秆还田时期地温低、秸秆降解慢,如何加速玉米秸秆低温腐解成为研究热点。【目的】从冷凉地区土壤中筛选具有高效降解纤维素能力的低温菌株,为秸秆的有效利用奠定基础。【方法】在低温培养条件下,采用稀释涂布平板法、羧甲基纤维素钠(sodium carboxymethyl cellulose,CMC-Na)水解圈测定法、胞外酶活测定法、秸秆失重法进行低温秸秆降解菌株的初筛、复筛和秸秆降解性能的测定;根据菌株形态学特征及ITSrDNA序列分析对筛选菌株进行鉴定;利用3,5-二硝基水杨酸(3,5-dinitrosalicylic acid,DNS)法和秸秆失重法对菌株在不同接种量、培养基初始pH、温度情况下的纤维素酶活力和玉米秸秆降解能力进行研究。【结果】以16°C为筛选温度,获得一株在刚果红-羧甲基纤维素钠平板上D/d值为2.17、CMC酶活力为703 U/mL的高产纤维素酶低温真菌SDF-25;该菌株在4°C可以生长,10-16°C为最适生长温度,37°C条件下仍能生长;综合菌株的形态学和分子生物学测定结果,菌株SDF-25为草酸青霉菌(Penicillium oxalicum);该菌株最佳产纤维素酶的培养条件为接种量2%、初始pH为7.0、培养温度为10°C,在该培养条件下菌株SDF-25的CMC酶活为993.3 U/mL。失重法测定接种SDF-25于10°C培养15 d时秸秆降解率为39.5%,16°C时为44.9%。【结论】草酸青霉菌SDF-25可在低温条件下生长并具有较强的纤维素酶生产能力,在秸秆还田方面具有良好的应用前景。