一株氨氧化细菌的分离鉴定及其氨氧化特性

以城市污水处理工程的好氧活性污泥为菌源,通过富集、分离纯化,筛选出一株自养型氨氧化细菌CM-NRO14,进一步研究了该菌株的系统发育地位及氨氧化特性。根据菌株的形态学特征、生理生化特性及16S rDNA序列测定分析,确定该菌株属于亚硝酸单胞杆菌。CM-NRO14菌株的最大氨氧化速率24.54 mg/(L·d),利用氨的Km值45.66 mg/L,氨自身的抑制常数2401 mg/L,最大氨氧化速率的氨浓度331.2 mg/L。亚硝酸盐对氨氧化的抑制常数1524 mg/L。氨氧化的最适pH 7.79。氨氧化的最适温度34℃。菌株在初始氨氮浓度为180 mg/L,最佳氨氧化pH、温度条件下,4 d内氨氮降解率达到91.6%,菌株倍增时间2.94 d。     

一株反硝化细菌的鉴定及其厌氧氨氧化能力的证明

从厌氧氨氧化反应器中分离获得了反硝化细菌D₃菌株. 综合其外部形态特性、生理生化特性、VitekGN检测结果、Biolog碳源利用特性, 以及菌株的(G+C)%(mol/mol)含量和系统发育分析, 将它归入门多萨假单胞菌(Pseudomonas mendocina). 该菌株是典型的反硝化细菌, 具有较强的反硝化活性, 当硝酸盐浓度为88.5 mg/L时, 反硝化速率最大, 为26.2 mg/(L·d). 最适生长pH为7.84, 最适生长温度为34.9℃. 该菌株显现较强的厌氧氨氧化能力, 对硝酸盐的最大利用速率为6.37 mg/(L·d), 对氨的最大利用速率为3.34 mg/(L·d), 消耗的氨氮和硝酸盐氮之比为1︰1.91. 该菌株可产生特殊的细胞结构, 与厌氧氨氧化密切相关, 推测这一特殊细胞结构可能是进行厌氧氨氧化的厌氧氨氧化体. 证明了反硝化细菌具有厌氧氨氧化活性, 扩大了厌氧氨氧化菌的种群范围, 为深入研究厌氧氨氧化菌及其在全球氮素循环中的贡献和进一步开发厌氧氨氧化工艺打下了良好的基础.     

硫代硫酸盐氧化菌TX的分离、鉴定及其生物学特性

从聚硫橡胶废水处理系统中分离到一株硫代硫酸盐氧化细菌TX.根据其形态学特征、生理特征和16S rRNA基因序列相似性分析,将该菌株初步鉴定为盐生硫杆菌属(Halothiobacillus sp.)(GenBank登录号为EU871645).该菌株能利用硫代硫酸盐、单质硫、连四硫酸盐、硫化物或亚硫酸盐为唯一能源进行自养生长,不能利用葡萄糖、蔗糖、果糖、乳糖、麦芽糖或酵母粉进行异养生长,为专性化能无机自养型硫杆菌.在以硫代硫酸钠为唯一能源的培养基中其最适生长温度为30℃～35℃,最适起始pH值为3.0～5.0.在矿物盐培养基中,硫代硫酸盐最终被氧化成硫酸,造成培养基pH持续下降.在摇瓶分批培养和硫胶废水处理过程中均检测到连四硫酸盐的积累,表明该菌株主要通过连四硫酸盐途径或"S4I"途径进行硫代硫酸盐的生物氧化.     

一株中度嗜热嗜酸铁氧化细菌特性研究*

从我国煤矿废石堆分离到一株中度嗜热嗜酸铁氧化细菌MLY菌株,最适生长温度50℃－54℃,最适pH1.2-1.4。MLY菌株是兼性化能自养菌,能利用酵母粉异养生长。在自养和混合营养条件下,能氧化Fe^2 、黄铁矿(FeS2)和元素硫(S^0)。自养营养时,氧化元素硫较弱。对比研究MLY菌株和氧化亚铁硫杆菌(Thiobacillus ferrooxidans)A10菌株对Fe^2 和黄铁矿的氧化作用,结果表明,MLY比A10的氧化速度快1倍多。     

一株新型同型产乙酸菌Clostridium sp.的自养和异养生长特性

【背景】同型产乙酸菌是一类利用乙酰辅酶A途径固定CO_2合成自身细胞物质并生成乙酸、乙醇等代谢产物的厌氧菌群,其分布广泛、种类繁多且代谢多样。深入研究同型产乙酸菌菌株的代谢能力及特性,对探索该种群的生理生化特性及其环境作用至关重要。【目的】研究一株同型产乙酸菌Clostridium sp. BXX的最适培养条件及其自养与异养生长特性。【方法】设置BXX菌株培养温度10-55°C、初始pH 6.0-9.0、NaCl浓度0-2.0%、不同氮源,测定菌体细胞含量和产物生成浓度,确定菌株最适培养条件。研究BXX菌株分别以H_2/CO_2、合成气、CO、葡萄糖、1,2-丙二醇、甲酸钠、乙二醇甲醚、甘油、丙酮酸和乳酸为底物时的底物消耗、产物生成、菌体细胞含量和pH等,探究其自养和异养生长特性。【结果】BXX菌株的最适培养温度为30°C,初始pH为7.0,NaCl浓度为1.0%,氮源为酵母粉。BXX菌株能以H2/CO2、合成气、葡萄糖、1,2-丙二醇、甲酸钠、乙二醇甲醚和甘油为底物生长,不能以CO、丙酮酸或乳酸为底物生长。【结论】BXX菌株既能自养生长产乙酸,又能异养生长产乙醇。BXX菌株是乙酸发酵的优良菌种资源,有较好的工业应用潜力。     

具抗菌活性海洋放线菌菌株JMC 06001的分离和鉴定

从湛江硇洲岛高盐泥样中分离到一株微嗜盐放线菌JMC 06001,该菌株的发酵产物具有很强的抗菌活性.菌株JMC 06001在多数培养基上生长良好,气生菌丝白色,基内菌丝淡黄色至浅棕色.在燕麦琼脂(ISP 3)、甘油-天门冬酰胺琼脂(ISP 5)、葡萄糖-天门冬酰胺琼脂及马铃薯浸汁琼脂上产生黄色可溶性色素,在酵母膏-麦芽膏琼脂(ISP2)、蛋白胨酵母膏铁盐琼脂(ISP6)和营养琼脂上产生棕色可溶性色素.生长温度范围为4℃～40℃,最适生长温度28℃.生长pH范围为6.0～9.0,最适pH 7.0.能在含0～1.5 mol/L NaCl的培养基上生长,最适生长盐浓度为0.2～0.5 mol/L NaCl.根据其形态学特征、生理生化特征、细胞化学分类特征和基于16S rRNA基因序列的系统发育分析结果,菌株JMC 06001被鉴定为链霉菌属(Streptomyces)的有效发表种波赛链霉菌(S.peucetius)的一个菌株.     

一株甲烷氧化菌的分离鉴定与特性

甲烷氧化菌是一类能以甲烷作为唯一碳源和能源进行同化和异化代谢的微生物。从若尔盖高原不同地点采集的样品中筛选得到一株名为XN1的甲烷氧化菌,根据此菌株的形态与16SrRNA序列同源性分析,证实该菌株属于Methylomonas属。对该菌株的培养条件进行研究的结果表明,以甲烷与甲醇共同作为碳源,硝酸钾和氯化铵共同为氮源时菌生长最好,最适生长温度为25℃,最适生长pH为6.5,培养基中CuSO4·5H2O和FeSO4·7H2O的浓度以0.03mg/L和0.4mg/L为宜。     

厌氧氨氧化菌富集培养物对羟胺的转化研究

【目的】羟胺是厌氧氨氧化的重要中间产物,本研究旨在探明厌氧氨氧化菌对羟胺的转化特性。【方法】采用厌氧氨氧化菌富集培养物,以羟胺和亚硝酸盐为基质进行分批培养试验,检测反应液中基质和产物的消涨情况。【结果】不接种厌氧氨氧化富集培养物时,羟胺和亚硝酸盐具有化学稳定性,彼此不发生化学反应;接种厌氧氨氧化富集培养物后,羟胺和亚硝酸盐发生化学反应;反应过程中有中间产物氨的产生和转化,最大氨氮积累浓度为0.338mmol/L;液相中总氮浓度从起始的4.694mmol/L降至结束时的0.812mmol/L,转化率为82.7%。羟胺和亚硝氮浓度均为2.5mmol/L时,羟胺最大比污泥转化速率为0.535mmol/(gVSS.h),是厌氧氨氧化反应体系中氨氮最大比污泥转化速率的1.81倍。将羟胺浓度提高至5.0mmol/L时,羟胺和亚硝氮转化速率分别提高26.7%和120.7%,最大氨氮积累浓度为0.795mmol/L;将亚硝氮浓度提高至5.0mmol/L时,羟胺和亚硝氮转化速率分别提高6.9%和9.0%,最大氨氮积累浓度为1.810mmol/L。【结论】厌氧氨氧化富集培养物能够转化羟胺,其对羟胺的转化速率高于对氨的转化速率。羟胺相对过量可显著加快羟胺和亚硝酸盐的转化速率,亚硝酸盐相对过量对羟胺和亚硝氮转化速率影响不大,提高羟胺或亚硝氮浓度均会增大中间产物氨氮的积累。实验现象可用van de Graaf模型解释,对于进一步开发厌氧氨氧化工艺具有重要的理论意义。     

在无有机碳源和缺氧条件下自养菌和异养菌脱氨氮作用

在无有机碳源和缺氧条件下,应用^15N示踪考察4株具有氨氧化作用的菌株在膜反应器中的氨氧化产气情况。当溶氧浓度（DO）〈0．5mg／L的缺氧条件下,自养菌Nitrosomonas sp．单株培养,能将6．3％的氨氮转化为氮气,^15N2产生量占氨氮消耗量的21．86％。自养菌株与异养菌株混合培养,可将30．86％的氨氮转化为氮气,^15N2产生量占氨氮消耗茸的80．38％。企无有机碳的条件下,自养菌和异养菌的协同代时作用,可大大提高缺氧氩氧化产氮气的效率。     

在无有机碳源和缺氧条件下自养菌和异养菌脱氨氮作用*

在无有机碳源和缺氧条件下,应用15N示踪考察4株具有氨氧化作用的菌株在膜反应器中的氨氧化产气情况。当溶氧浓度(DO)<0.5mg/L的缺氧条件下,自养菌Nitro-som onas sp.单株培养,能将6.3%的氨氮转化为氮气,¹⁵N₂产生量占氨氮消耗量的21.86%。自养菌株与异养菌株混合培养,可将30.86%的氨氮转化为氮气,¹⁵N₂产生量占氨氮消耗量的80.38%。在无有机碳的条件下,自养菌和异养菌     

Modeling and simulation of oxygen-limited partial nitritation in a membrane-assisted bioreactor (MBR)

Combination of a partial nitritation process and an anaerobic ammonium oxidation process for the treatment of sludge reject water has some general cost-efficient advantages compared to nitrification-denitrification. The integrated process features two-stage autotrophic conversion of ammonium via nitrite to dinitrogen gas with lower demand for oxygen and no external carbon requirement. A nitrifying membrane-assisted bioreactor (MBR) for the treatment of sludge reject water was operated under continuous aeration at low dissolved oxygen (DO) concentrations with the purpose of generating nitrite accumulation. Microfiltration was applied to allow a high sludge retention time (SRT), resulting in a stable partial nitritation process. During start-up of the MBR, oxygen-limited conditions were induced by increasing the ammonium loading rate and decreasing the oxygen transfer. At a loading rate of 0.9 kg N m(-3) d(-1) and an oxygen concentration below 0.1 mg DO L(-1), conversion to nitrite was close to 50% of the incoming ammonium, thereby yielding an optimal effluent within the stoichiometric requirements for subsequent anaerobic ammonium oxidation. A mathematical model for ammonium oxidation to nitrite and nitrite oxidation to nitrate was developed to describe the oxygen-limited partial nitritation process within the MBR. The model was calibrated with in situ determinations of kinetic parameters for microbial growth, reflecting the intrinsic characteristics of the ammonium oxidizing growth system at limited oxygen availability and high sludge age. The oxygen transfer coefficient (K(L)a) and the ammonium-loading rate were shown to be the appropriate operational variables to describe the experimental data accurately. The validated model was used for further steady state simulation under different operational conditions of hydraulic retention time (HRT), K(L)a, temperature and SRT, with the intention to support optimized process design. Simulation results indicated that stable nitrite production from sludge reject water was feasible with this process even at a relatively low temperature of 20 degrees C with HRT down to 0.25 days.     

新异养硝化菌Colloides sp.JZ1-1的鉴定及其硝化性能

从驯化后的活性污泥中筛分、诱变出一株性能较好的异养硝化菌JZ1-1.经形态及生理生化特性分析,鉴定菌株JZ1-1为胶样菌属(Colloides sp.).分别考察了碳源、C/N、pH、溶解氧、温度和铵态氮初始浓度对JZ1-1硝化性能的影响.结果表明:菌株对柠檬酸钠的利用较好;C/N为10 ～14、30℃、pH 6-9和转速150 r·min-1以上有利于铵态氮的降解;菌株对中高浓度铵态氮废水(100 mg·L-1≤铵态氮浓度≤500 mg·L-1)的降解效果显著.经5次继代培养,菌株的稳定性较好.     

A rapid and simple respirometric biosensor with immobilized cells of Nitrosomonas europaea for detecting inhibitors of ammonia oxidation

As obligate chemolithotrophs, ammonia-oxidizing bacteria (AOB) grow very slowly and are known to be extremely sensitive to a wide variety of inhibitors. Since it is generally accepted that inhibition of ammonia oxidation by AOB results in a total failure of nitrogen removal, it is necessary to develop a method to detect inhibitors of ammonia oxidation in wastewater. Since ammonia oxidation accompanies oxygen consumption, ammonia oxidation can be easily evaluated by measuring oxygen consumption rate using a dissolved oxygen (DO) probe. In this study, a rapid and simple respirometric biosensor using the pure culture of Nitrosomonas europaea was developed. N. europaea was cultivated in a continuous fermentor operating at the dilution rate of 0.008 h(-1) to obtain physiologically constant cells and was immobilized onto the dialysis membrane through filtration. DO, determined by the biosensor, started to increase 30 s later after ammonia oxidation inhibitor was fed, and a new steady-state DO was obtained in 10-30 min. For this DO profile, steady-state kinetics was applied to evaluate ammonia oxidation efficiency. The concentration of a toxic compound causing 50% decrease of oxygen-consumption activity (EC50) was determined for different chemicals. The EC50 values obtained with the biosensor (0.018 mg l(-1) for allylthiourea, 0.027 mg l(-1) for thioacetamide, 1.10 mg l(-1) for phenol and 0.0 1mg l(-1) for thiourea) indicated that the developed biosensor was highly sensitive to a variety of the inhibitors. It was also shown that the biosensor is applicable for on-line real time monitoring.     

Growth of Nitrosomonas europaea on hydroxylamine

Abstract Hydroxylamine is an intermediate in the oxidation of ammonia to nitrite, but until now it has not been possible to grow Nitrosomonas europaea on hydroxylamine. This study demonstrates that cells of N. europaea are capable of growing mixotrophically on ammonia and hydroxylamine. The molar growth yield on hydroxylamine (4.74 g mol⁻¹ at a growth rate of 0.03 h⁻¹) was higher than expected. Aerobically growing cells of N. europaea oxidized ammonia to nitrite with little loss of inorganic nitrogen, while significant inorganic nitrogen losses occurred when cells were growing mixotrophically on ammonia and hydroxylamine. In the absence of oxygen, hydroxylamine was oxidized with nitrite as electron acceptor, while nitrous oxide was produced. Anaerobic growth of N. europaea on ammonium, hydroxylamine and nitrite could not be observed at growth rates of 0.03 h⁻¹ and 0.01 h⁻¹.     

Environmental factors influencing the rate of hydrocarbon oxidation in temperate lakes.

Rates of hydrocarbon biodegradation were estimated by following oxygen uptake during mineral oil oxidation or oxidation of [1-14C]hexadecane to 14CO2, when these substrates were added to natural water samples from Wisconsin lakes. A lag phase preceded hydrocarbon oxidation, the length of which depended on population density or on factors influencing growth rate and on the presence of nonhydrocarbon organic compounds. Hydrocarbon oxidation was coincident with growth and presumably represented the development of indigenous hydrocarbon-degrading microorganisms in response to hydrocarbon additions. In detailed studies in Lake Mendota, it was found that, despite the continued presence of hydrocarbon-degrading microorganisms in water samples, seasonal variations in the rates of mineral oil and hexadecane oxidation occurred which correlated with seasonal changes in temperature and dissolved inorganic nitrogen and phosphorus. The temperature optimum for oil biodegradation remained at 20 to 25 C throughout the year, so that temperature was the main limiting factor during winter, spring, and fall. During summer, when temperatures were optimal, nutrient deficiencies limited oil biodegradation, and higher rates could be obtained by addition of nitrogen and phosphorus. The rates of hydrocarbon biodegradation were thus high only for about 1 month of the ice-free period, when temperature and nutrient supply were optimal. Nutrient limitation of oil biodegradation was also demonstrated in 25 nutrient-poor lakes of northern Wisconsin, although in almost every case oil-degrading bacteria were detected. Knowledge of temperature and nutrient limitations thus will help in predicting the fate of hydrocarbon pollutants in freshwater.     

Environmental factors influencing the rate of hydrocarbon oxidation in temperate lakes.

Rates of hydrocarbon biodegradation were estimated by following oxygen uptake during mineral oil oxidation or oxidation of [1-14C]hexadecane to 14CO2, when these substrates were added to natural water samples from Wisconsin lakes. A lag phase preceded hydrocarbon oxidation, the length of which depended on population density or on factors influencing growth rate and on the presence of nonhydrocarbon organic compounds. Hydrocarbon oxidation was coincident with growth and presumably represented the development of indigenous hydrocarbon-degrading microorganisms in response to hydrocarbon additions. In detailed studies in Lake Mendota, it was found that, despite the continued presence of hydrocarbon-degrading microorganisms in water samples, seasonal variations in the rates of mineral oil and hexadecane oxidation occurred which correlated with seasonal changes in temperature and dissolved inorganic nitrogen and phosphorus. The temperature optimum for oil biodegradation remained at 20 to 25 C throughout the year, so that temperature was the main limiting factor during winter, spring, and fall. During summer, when temperatures were optimal, nutrient deficiencies limited oil biodegradation, and higher rates could be obtained by addition of nitrogen and phosphorus. The rates of hydrocarbon biodegradation were thus high only for about 1 month of the ice-free period, when temperature and nutrient supply were optimal. Nutrient limitation of oil biodegradation was also demonstrated in 25 nutrient-poor lakes of northern Wisconsin, although in almost every case oil-degrading bacteria were detected. Knowledge of temperature and nutrient limitations thus will help in predicting the fate of hydrocarbon pollutants in freshwater.     

氮源种类及溶氧水平对锁掷酵母产类胡萝卜素组分的影响

在锁掷酵母（Sporidioboluspararoseus）发酵产类胡萝卜紊的过程中,发酵产物中类胡萝卜紊种类繁多,而且性质相似,加大了不同色素分离纯化的难度。为定向积累不同种类的类胡萝卜素,以本实验室保藏锁挪酵母JD-2为出发菌,研究了氮源种类和浓度及溶氧对锁掷酵母产类胡萝卜素的影响,并在7L发酵罐中进行了补料分批发酵试验。发现培养基中同时添加有机氮源和无机氮源且溶氧控制较低（5％）时有利于β-胡萝卜素的大量积累,最佳有机氮源和无机氮源分别为玉米浆（20g／L）、硫酸铵（5g／L）。补料分批发酵时β-胡萝卜素产量达到31．28mg／L,红酵母烯12．38mg／L。培养基中只添加有机氮源且相对溶氧控制相对较高（30％）时有利于红酵母烯的大量积累,最佳有机氮源为酵母膏（20g／L）。补料分批发酵时红酵母烯产量达到38．96mg／L,8．胡萝卜素12．36mg／L。     

可溶性TRAIL蛋白的高密度培养及补料策略研究

采用分批补料的方法高密度培养重组大肠杆菌C600/PbvTRAIL制备人可溶性TRAIL蛋白,优化发酵工艺,探索简单高效的分离纯化方法并测定蛋白生物活性。通过比较几种不同的补料策略：间歇流加、Dostat、pHstat,摸索了一种流加策略,即DOstatpHstat组合流加,有效的避免了发酵过程中,尤其是诱导表达阶段乙酸积累的增加,使TRAIL蛋白在高密度培养条件下,得到高效表达。菌体密度最终达到300g/L（WCW）以上,可溶性TRAIL蛋白占菌体总蛋白的4.2%,含量为1.1g/L。在整个发酵过程中,乙酸浓度接近于0,且未使用任何特殊手段,如纯氧、加压等,简化了发酵工艺,降低了发酵成本,为TRAIL的工业化生产创造了条件。     

Simultaneous NH3 oxidation and N2 production at reduced O2 tensions by sewage sludge subcultured with chemolithotrophic medium

The ammonia oxidation rate by sewage sludge was determined as a function of the dissolved oxygen tension. Samples of sludge were taken from a domestic waste water treatment pilot plant in which sludge was completely retained by membrane filtration. The samples were subcultured chemolithotrophically in recycling reactors. The gas supplied was a mixture of pure argon and oxygen. The K _O2 for ammonia oxidation was estimated to be 0.97 (±0.16) kPa dissolved oxygen. Together with ammonia oxidation and oxygen consumption, dinitrogen gas was produced. So, aerobic denitrification occurred. At dissolved oxygen tensions of 1.25 kPa and higher, the dinitrogen production rate (per N-mole) equalled 20% of the ammonia oxidation rate. This proportion was even 58% at 0.3 kPa dissolved oxygen. At 0.15 kPa dissolved oxygen, however, nitrification hardly proceeded, while dinitrogen production soon stopped. Most likely, a nitrifier concomitantly oxidized ammonia and reduced nitrite to dinitrogen.     

Physiology and morphology of Legionella pneumophila in continuous culture at low oxygen concentration.

Two strains of Legionella pneumophila serogroup 1 monoclonal subgroup Pontiac were grown for the first time in continuous culture using a chemically defined medium. The influence of temperature on physiology and morphology was investigated by fixing the growth rate (equal to the dilution rate, D) at 0.08 h-1 and controlling the pH and dissolved oxygen concentration of the culture. Serine provided the principal source of carbon and energy but growth was limited by tyrosine. The bacterium behaved as a microaerophile in this medium, with maximal growth occurring at 0.31 (mg O2)I-1 (equivalent to a dissolved oxygen tension of 4% (v/v) air saturation at 30 degrees C). The cultures consisted of flagellated, short rods at 24 degrees C, but exhibited an increased level of pleomorphism and the loss of flagella as the temperature was increased to 37 degrees C. The presence of intracellular granules was noted, and their abundance was temperature-dependent. Polyhydroxybutyrate was present in L. pneumophila, and the proportion of the cell dry weight that it accounted for varied with temperature, being maximal at 24 degrees C. The ratio of saturated to unsaturated fatty acids in the cells decreased as the temperature was reduced towards 24 degrees C, so as to maintain membrane fluidity at low growth temperature.