海洋沉积物中一株铁还原细菌分离及Fe(Ⅲ)还原性质

以渤海(塘沽海域)沉积物为材料,富集异化铁还原混合菌群。采用三层平板方法,从混合菌群中纯化出一株异化铁还原细菌KB52。通过形态观察和16S r RNA基因序列分析,菌株命名为Klebsiella sp.KB52(Gene Bank号KM233642)。在葡萄糖为电子供体,Fe(OH)_3为电子受体条件下厌氧培养菌株KB52,其细胞生长和Fe(Ⅲ)还原具有明显耦合关系。碳源分别设置为葡萄糖、乳酸钠、丙酮酸钠、乙酸钠、甲酸钠和丙酸钠,在海水培养条件下菌株KB52以丙酮酸钠为碳源时,菌株培养液累积Fe(Ⅱ)浓度最高,为4.41 mmol/L±0.59 mmol/L。菌株KB52在设定NaCl浓度范围内,都能够生长并具有铁还原性质,菌株表现出较强的耐盐性。NaCl质量浓度为4 g/L时,菌株KB52还原Fe(Ⅲ)效率最高,Fe(Ⅱ)达到4.95 mmol/L±0.72 mmol/L。铁还原细菌KB52在淡水和海水条件下能够生长并具有铁还原性质,可用于近海沉积物中微生物介导异化Fe(Ⅲ)还原过程,进一步应用于治理海洋环境污染。     

辽河口湿地耐盐石油降解菌作用效果与条件优化研究

采用投加过氧化氢、吐温-80和活性炭纤维等方法对筛选、分离自辽河口湿地石油污染土壤样品的耐盐高效石油降解菌的优化条件进行研究。结果表明,过氧化氢的最佳投加量为150mg/L;吐温-80的最佳投加量为160mg/L;活性炭纤维的最佳投加量为6g/L。3种优化条件下石油降解率均有增加,培养1周后,耐盐菌株在10℃下的石油降解率在40%以上,而在最适温度30℃下的石油降解率达到80%以上。     

海水养殖环境中拟除虫菊酯类农药降解菌的分离鉴定及降解特性研究

从近岸海水养殖环境中分离获得一株能高效降解氯氰菊酯(Cypermethrin,CYP)和溴氰菊酯(Deltamethrin,DEL)的菌株HS-10,经生理生化和16S rDNA分析,初步鉴定其为假交替单胞菌(Pseudoalteromonas sp.)。不同降解条件下的实验结果表明,菌株HS-10在pH 7.0、温度28°C的环境中具有较好的生长和降解效率,在该条件下,对初始浓度为100mg/L的CYP和DEL的降解效率分别为75.6%和90.9%。进一步采用发光细菌和核磁共振(NMR)方法对菌株HS-10的降解产物进行分析,结果表明,CYP和DEL主要通过微生物降解消除,其降解产物的毒性显著降低。同时,考察了菌株HS-10对氯氰菊酯、溴氰菊酯、联苯菊酯(BIF)、氟氯氰菊酯(CYF)和氰戊菊酯(FEN)5种拟除虫菊酯类农药(浓度分别为100mg/L)的降解效率,结果表明,对5种农药的降解率均达到50%以上,本研究获得的菌株HS-10可用于海水养殖环境中拟除虫菊酯类农药残留污染的生物修复。     

高效硝化与反硝化功能菌株的分离筛选及其性能研究

本研究利用选择培养基从鳗鲡精养殖池和循环水水处理系统中分离筛选出二株脱氮细菌,应用16S rRNA分子生物学鉴定,确定菌株的种属。菌株NB-1属于芽孢杆菌属（Bacillus）、菌株DB-1是恶臭假单胞菌（Pseudomonas putida）。对菌株硝化及反硝化作用性能的研究结果表明：菌株NB-1具有良好的亚硝化及硝化作用性能,在投菌量为0.025%时,氨氮降解率为69.5%,亚硝酸盐氮降解率为99.2%。菌株DB-1在厌氧条件下表现出良好的反硝化作用性能,最适投菌量为0.6%,最佳C/N为12,对硝酸盐氮的降解率为94.6%,对总氮的降解率为64.0%。因此本研究筛选出的二株脱氮细菌可以广泛应用于养殖水体水质调控,具有良好的市场应用前景。     

国产沸石的改性处理及其在海水硝酸盐氮同位素预处理中的应用

使用多种改性方法对几种国产天然沸石进行改性处理,提高其铵氮吸附率,制备符合海水硝酸盐氮同位素预处理要求的沸石。发现重力筛选可提高沸石铵氮吸附率16%以上,钠改性及酸改性后钠改性可提高沸石铵氮吸附率80%以上,微波改性和超声波改性均可进一步提高沸石铵氮吸附率。改性处理后,几种沸石在酸性条件下对低浓度铵氮吸附率达90%以上,其氮同位素分馏较美国UOP沸石分馏系数更小,且更稳定。改性后的国产沸石更适于海水硝酸盐氮同位素预处理。应用改性后沸石对长江口海域硝酸盐水样进行了分析,结果表明,改性后沸石可以应用于海水中溶解态硝酸盐的氮同位素分析,为海水中溶解态氮的来源问题及循环机理研究等提供了有效信息。     

北戴河滨海湿地根际石油降解菌的筛选及功能分析

以北戴河碱蓬-芦苇滨海湿地采集的5个根际土壤样品为研究材料,使用以原油为唯一碳源的选择培养基富集培养石油降解菌,采用重量法评价降解效率,通过GC-MS分析研究高效降解菌的降解特性,利用排油圈法测定降解菌的表面活性剂产生能力。研究结果表明,初筛获得22株石油降解菌,在25℃降解21d的条件下对石油的降解率为17%～38%,其中qhd2B降解率最高,为38%,其中8株具有较好的产表面活性剂的能力。16s测序结果经BLAST比对,所分离获得的22株菌中,8株菌属于放线菌门(Actinobacteria),13株菌属于变形菌门(Proteobacteria),有一株拟杆菌门(Bacteroidetes)丽水菌属(Yeosuana)。选取石油降解率在31%以上菌株进行GC-MS分析,结果显示其对大部分烷烃和多环芳香烃都有较好的降解效果。其中菌株qhd2B对烷烃和芳烃的总降解率分别为79.9%和76.7%,是一株优良的石油降解菌。     

抗硅藻附着活性细菌的筛选、鉴定与发酵条件优化

从美国华盛顿州圣璜岛海域的8 种海绵样品的120 株共附生微生物中, 筛选出11 株细菌的粗提物能有效抑制硅藻附着。其中683 号菌株活性最高, 对5 种受试硅藻的平均抑制率达到98.0%。结合形态学特征和扫描电镜观察, 以及16S rDNA 序列分析, 鉴定该菌株为短小芽孢杆菌Bacillus pumilus。并采用正交试验对该菌株进行发酵条件优化, 结合粗提物的产量和抗硅藻附着活性进行统计学分析,确定了该菌株的最佳发酵条件是: 发酵温度25℃, pH 7.5, NaCl 浓度19.45 g/L, 蛋白胨浓度5 g/L。     

自组装膜进样质谱系统及其在砂质沉积物异化硝酸盐还原研究中的应用

沉积物中的异化硝酸盐还原过程对于海洋氮循环起着至关重要的作用。基于15N标记的培养技术是目前测定沉积物异化硝酸盐还原的主要手段。准确快速测定15N标记的产物(29N2、 30N2)是量化异化硝酸盐还原各个过程速率的关键。本研究自行组装膜进样质谱系统用于29N2和30N2的测定,对其测量条件进行了优化。结果表明,进样蠕动泵进样流速0.80 mL/min,进样时间3~3.5 min,恒温槽温度20~25℃,同时铜还原炉温度在300~600℃的条件下,^29N2/^28N2和^30N2/^28N2的测试精密度分别可以控制在0.1%和1%以内,比较适合29N2和30N2的测定。利用自组装的膜进样质谱系统结合15N标记的培养技术研究了青岛石老人沙滩沉积物中的异化硝酸盐还原过程。石老人沙滩沉积物不存在将硝酸盐完全还原为氮气好氧的反硝化。厌氧铵氧化、厌氧反硝化和异化硝酸盐还原为铵(Dissimilatory Nitrate Reduction to Ammonium,DNRA)的潜在速率(以湿沉积物N计)分别为(0.05±0.01) nmol/(cm^3·h),(2.32±0.21) nmol/(cm^3·h)和(1.02±0.15) nmol/(cm^3·h)。厌氧反硝化是硝酸盐异化还原主要的贡献者,其比例接近70%,其次是DNRA,比例可达30%,而厌氧铵氧化的贡献最低,仅为1%。在N2产生过程中,主要贡献者是反硝化,厌氧铵氧化的贡献仅为2%。     

长江口可培养耐(嗜)盐细菌群落结构研究

针对长江口10个站位样品,采用HM培养基和ZMCA培养基分离培养耐(嗜)盐菌,通过PCR方法获取156株细菌的16S rDNA序列。其中,由HM培养基分离获得136株菌株,可划分为33个分类单元(OUT),分属α-变形菌纲(Alphaproteobacteria)、γ-变形菌纲(Gammaproteobacteria)和厚壁菌门(Firmicutes)的芽孢杆菌纲(Bacilli)3个类群,其中γ-变形菌纲为优势耐(嗜)盐细菌;靠近海岸的站点M5-1,N6-2和M4-1耐(嗜)盐细菌均属于厚壁菌门,而远离海岸的站点耐(嗜)盐细菌均属于变形菌门。以ZMCA培养基分离获得的20株菌株分属α-变形菌纲和放线菌纲(Actinobacteria),可划分为10个OTU。由HM培养基分离获得的菌株中,70.5%属于耐盐菌,29.5%属于中度嗜盐菌;由ZMCA培养基分离获得的菌株均为耐盐菌。HM培养基分离得到的136株细菌的硝酸盐和亚硝酸盐还原性分析表明,亚硝酸盐还原菌数量仅为硝酸盐还原菌的1/3左右。     

循环水养殖系统中好氧反硝化细菌的分离和应用

为了分离好氧反硝化细菌,探究其好氧反硝化过程。利用BTB培养基,从珍珠龙胆(♀Epinephelus fuscoguttatus×♂Epinephelus lanceolatu)循环水养殖系统的生物滤池中筛选出具有硝酸盐去除能力的细菌,并选择脱氮效果良好的菌株进行好氧反硝化反应器的构建,开展反硝化应用研究。本研究共分离出8株具有去除硝酸盐能力的菌株,经反硝化性能测定,都可大幅去除硝酸盐,同时也存在不同程度的氨氮和亚硝酸盐的积累;选择Z1、Z8两株脱氮效果较好的菌株进行好氧反硝化反应器的混合接种试验,结果显示反应器挂膜迅速、高效,接种2周后即达到相对稳定的水处理状态,硝酸盐去除率超过98.8%(约0.827g NO-3-N/(m2·d)),总氮去除率超过71.8%(约0.687g TN/(m2·d)),亚硝酸盐和氨氮的积累不明显,脱氮效果良好。     

氮浓度对四株海洋绿藻总脂含量和脂肪酸组成的影响

本文用NaNO3作为氮源,分别作了氮浓度的五个水平对2属(小球藻属、裂丝藻属)的四株绿藻的生长率、总脂含量及主要脂肪酸组成的影响。C19、C97和C102的脂肪含量随N浓度的改变而有较大变化,其中C102在1.6m mol/L时脂肪含量达到最大值(24.8%)。C95的脂肪含量随氮浓度变化不大。同时,由氮浓度引起的平均生长率μ与总脂含量之间无明显相关关系。四株绿藻的EPA(20∶5n-3)和PUFAs(polyunsaturated fatty acids)含量随培养基中氮浓度的改变有较大变化但因种而异。C95和C97均在中等氮浓度时EPA含量达到最大值,分别为23.8%和27.4%。C19和C102在高氮浓度(40mmol/L)获得EPA的最大值,分别为25.7%和26.6%。     

化学发光法测定天然水体中低浓度硝酸盐和亚硝酸盐的含量

传统分光光度法测定硝酸盐和亚硝酸盐含量时检测限较高,无法测定部分海洋表层及寡营养盐海域低含量的硝酸盐和亚硝酸盐浓度,急需采用一种新的测定低浓度硝酸盐和亚硝酸盐的方法.化学发光法用于测定水体中硝酸盐和亚硝酸盐含量,具有灵敏度高、检测限低、样品用量少,不受悬浮颗粒物、有色物质影响及样品批量测定等优点.本文通过探索载气流速、...     

纳米氧化锌和菲对长江口附近海域沉积物反硝化作用及功能菌群落结构的影响

本研究在长江口附近海域采集表层沉积物,采用实验室模拟培养与分子生物学手段相结合的方法,通过测定纳米氧化锌(ZnO NPs)和菲(Phe)胁迫下沉积物中NO^-₃-N和NO^-₂-N浓度和反硝化还原酶活性及反硝化细菌基因丰度和群落多样性变化,目的是比较研究ZnO NPs和Phe对河口区沉积物反硝化作用及功能菌群落结构的影响,并探讨其作用过程和可能的作用机制。结果表明:ZnO NPs和Phe对沉积物硝酸盐还原能力和亚硝酸还原能力均产生抑制作用,浓度越高,抑制作用越强,其中亚硝酸盐还原过程受到2种污染物抑制更强烈,加重了沉积物亚硝酸盐的累积。ZnO NPs对沉积物硝酸盐还原能力、硝酸还原酶活性、narG基因丰度的抑制程度大于Phe,Phe对沉积物亚硝酸盐还原能力、亚硝酸还原酶和nirS基因丰度的抑制程度大于ZnO NPs,表明对反硝化还原酶和反硝化功能基因的抑制是外源污染物胁迫影响反硝化过程的主要机制。ZnO NPs和Phe降低了沉积物反硝化菌群落多样性水平,增加沉积物中Halomonas的优势度,降低了Bacillus的优势度,但Phe对沉积物群落多样性和组成的影响更加明显,说明Phe对长江口海区的生态影响大于ZnO-NPs。     

A sensor package for the simultaneous determination of nanomolar concentrations of nitrite, nitrate, and ammonia in seawater by fluorescence detection

A fluorescence-based chemistry has been developed for the detection of nitrite and nitrate (as excess nitrite following reduction of nitrate to nitrite). Detection limits are 4.6 and 6.9 nM, respectively. The technique capitalizes on the triple bond between the two nitrogen atoms within the diazonium ion formed via the well-known reaction between an acidified nitrite sample and an aromatic primary amine. Fluorescence of π-electrons within this bond allows this reaction to be probed with standard fluorescence spectroscopy. Reverse Flow Injection Analysis (rFIA) is used to correct for background fluorescence from leachates and naturally occurring dissolved organic matter (DOM). Comparisons of samples analyzed for nitrite with this technique and with a highly-sensitive chemiluminescent method [Braman, R.S., Hendrix, S.A., 1989. Nanogram nitrite and nitrate determination in environmental and biological materials by vanadium (III) reduction with chemiluminescence detection. Analytical Chemistry, 61 (24) 2716–2718] showed excellent agreement between the two methods (slope=0.9996 and r²=0.9956). These fluorescent nitrite and nitrate + nitrite chemistries were coupled in a sensor package with a modified version of a fluorescent ammonia chemistry [Jones, R.D., 1991. An improved fluorescence method for the determination of nanomolar concentrations of ammonia in natural waters, Limnology and Oceanography. 36(4) 814–819], which also has a nanomolar detection limit. The throughput rate of the fully automated three-channel instrumentation is 18 samples per hour. A field experiment demonstrated the capability of the nutrient sensor package to determine horizontal gradients in nitrate, nitrite, and ammonia in oligotrophic surface waters.     

A chemiluminescent technique for the determination of nanomolar concentrations of nitrate and nitrite in seawater

A chemiluminescent analysis technique for the determination of nanomolar quantities of nitrate, nitrate plus nitrite or nitrite alone in seawater is described. The method depends on the selective reduction of these species to nitric oxide which is then determined by its chemiluminescent reaction with ozone, using a commercial nitrogen oxides analyzer. The necessary equipment is compact and sufficiently sturdy to allow shipboard use. A precision of ±2 nM is claimed with analytical rates of 10–12 samples h^?1, and modifications are discussed to allow doubling the analytical rate.     

Nitrogen Uptake in Permanently Well-mixed Temperate Coastal Waters

Uptake rates of ammonium, nitrate, urea and nitrite were measured for 1 year (1988) at a coastal station in the well-mixed waters of the western English Channel. Ammonium was the major form of nitrogen (N) utilized (48%) by phytoplankton, followed by nitrate (32%), urea (13%) and nitrite (7%). Seasonal changes of uptake of ammonium, nitrate and urea showed a broad, intense summer maximum. Nitrite uptake was low throughout the year except for a peak value in June. Uptake rates of ammonium and nitrate were independent of substrate concentrations, whereas those of urea and nitrite were not. The summer maxima of ammonium, nitrate and total N uptake, and the significant relationships of N-uptake index to ambient light, and of chlorophyll-a-specific N uptake to surface-incident light, indicate that light is the major factor controlling N uptake in these waters. This is due to the permanent vertical mixing which reduces the mean light available for N uptake to <15% of the incident light. Mixing also injects regenerated N continuously into the euphotic zone, thus alleviating nitrogen limitation and accounting for the larger proportion of regenerated N uptake in total N uptake.     

别样玫瑰变色杆菌(Aliiroseovarius sp.)Z3基因组测序及比较基因组分析

菌株Z3是一株从凡纳滨对虾(Litopenaeus vannamei)肠道样品中分离出的细菌。本研究利用Illumina HiSeq测序平台对其进行了测序, 用SOAPdenovo等软件进行基因组组装、系统发育分析、基因预测和功能注释, 并与别样玫瑰变色杆菌属(Aliiroseovarius)已知的5个种的模式株进行了比较基因组分析。基因组注释结果显示, Z3基因组大小为3525503bp, GC(guanylate and cytidylic acid)含量为59.4%, 预测包含3509个编码蛋白基因。通过16S rRNA基因全长序列比对、平均核苷酸一致性(ANI)、DNA-DNA杂交(DDH)和共线性分析发现, 菌株Z3与Aliiroseovarius crassostreae CV919-312T的16S rRNA基因全长序列相似度最高, 为98.20%, 与Aliiroseovarius sediminilitoris DSM 29439T的DDH和ANI值最高, 分别为26.80%和84.95%, 属于Alliroseovarius属的新种, 将其命名为Aliiroseovarius sp. Z3。经比较基因组分析发现, Aliiroseovarius sp. Z3与5个近缘的模式株细菌共享2005个直系同源核心基因簇; Z3拥有的413个独立基因经注释, 主要与碳水化合物转运与代谢、氨基酸转运与代谢、复制、重组及修复等功能相关。功能注释发现Z3具有进行完整的反硝化途径的相关基因, 其利用的可能是环境中的硝酸盐、亚硝酸盐等。本研究对Z3全基因序列的注释、功能和比较基因组的分析, 不仅丰富了Aliiroseovarius属细菌基因组资源, 还为深入研究其反硝化特性等提供了基础。     

Fine scale distribution of nitrous oxide in marine sediments

Vertical profiles of nitrous oxide and other inorganic nitrogen compounds in the sediments in Tokyo Bay and its vicinity were measured using the whole core squeezing method. Within the bay area, vertical profiles of nitrous oxide in the interstitial waters showed one or two distinctive peaks around the depth of 2–4 cm, which corresponded to the peaks of nitrite and nitrate. In situ formation of nitrous oxide through nitrification was suggested in those sediments, which was possibly activated by the presence of benthic animals. On the other hand, at the deep sediment off Tokyo Bay the profiles of nitrous oxide, nitrite and nitrate gave a monotonous single peak, indicating less bioturbated condition. Denitrification or dissimilatory nitrate reduction might be important for the formation of nitrous oxide peaks in the latter type of sediment.