南极冰藻绿藻B-7的异养培养

2001年10月至2002年4月在中国第18次南极科学考察时采得南极海水水样和海冰样品,从中分离纯化出南极冰藻绿藻B 7,通过单因子试验及L25(56)正交试验表对其进行异养培养条件研究,并采用全量凯氏定氮法及气相色谱法测定该藻在异养培养条件下生化组成的变化。结果表明,在异养培养条件下,绿藻B 7的生长速率约为自养条件下的2倍。绿藻B 7异养培养最适有机碳、氮源分别为葡萄糖、蛋白胨;绿藻B 7的最佳异养培养条件为:培养温度4℃,培养液起始pH值6～8,250mL三角瓶装液量50mL,接种量20%。与自养条件相比,异养绿藻B 7细胞内生化组成有所变化:蛋白质含量升高,饱和脂肪酸及多不饱和脂肪酸占总脂肪酸的含量下降,单不饱和脂肪酸含量基本维持不变或有所上升。研究说明,通过异养培养可有效地提高绿藻B 7的藻体最终收获量,且异养条件下绿藻B 7的营养价值不低于自养绿藻B 7。在我国北方寒冷季节,可以利用异养培养方式大规模培养南极绿藻B 7,为育苗提供充足的饵料。     

杂色鲍健康苗种与患病苗种异养细菌致病毒力因子的比较研究

2002年以来,南方杂色鲍苗出现大规模死亡的问题,严重影响到南方鲍产业的发展。为寻找病因,于2004年自广东深圳下沙鲍鱼场培苗过程中变白掉板的杂色鲍苗以及官湖养殖场健康鲍苗各分离到19株和15株异养细菌,在此基础上研究了它们蛋白酶、淀粉酶、明胶酶、磷脂酶的产酶能力及溶血现象,并对它们的产酶能力及溶血能力进行了比较分析。结果表明,虽然来自下沙病鲍苗的菌株比来自官湖的健康鲍苗的溶血能力差;但它们大多数具有较强的分泌蛋白酶、明胶酶、淀粉酶、磷脂酶的能力。考虑到胞外酶是细菌的毒力因子之一,据此推测,下沙鲍鱼场鲍苗发生脱板死亡的原因可能与这些菌株能分泌胞外酶有关。     

Root exclusion methods for partitioning of soil respiration: Review and methodological considerations

Soil respiration is a vital process in all terrestrial ecosystems, through which the soil releases carbon dioxide (CO₂) into the atmosphere at an estimated annual rate of 68-101 Pg carbon, making it the second highest terrestrial contributor to carbon fluxes. Since soil respiration consists of autotrophic and heterotrophic constituents, methods for accurately determining the contribution of each constituent to the total soil respiration are critical for understanding their differential responses to environmental factors and aiding the reduction of CO₂ emissions. Owing to its low cost and simplicity, the root exclusion (RE) technique, combined with manual chamber measurements, is frequently used in field studies of soil respiration partitioning. Nevertheless, RE treatments alter the soil environment, leading to potential bias in respiration measurements. This review aims to elucidate the current understanding of RE, i.e., trenching (Tr) and deep collar (DC) insertion techniques, by examining soil respiration partitioning studies performed in several ecosystems. Additionally, we discuss methodological considerations when using RE and the combinations of RE with stable isotopic and modeling approaches. Finally, future research directions for improving the Tr and DC insertion methods in RE are suggested.     

印度西部卡奇沼泽地沉积物中微生物活性和人工培养细菌多样性的研究

The Great Rann of Kachchh, a vast expanse of salt desert in Western India is a unique hostile ecosystem posing an extreme environment to life forms due to high salt content, hyper-axid climate, seasonal water logging and extremes of temperature. In the virtual absence of natural vegetation, soils and sediments of Rann of Kachchh axe microbially dominated ecosystems. In the present study microbial activity and the diversity of cultivated heterotrophic bacteria were investigated in the sediments collected along a 5-m exposed section at Khadir Island in the Great Rann of Kachchh. Microbial activity （as an index of sediment enzymes） was found to be high in the middle of the section （200-280 cm）. Dehydrogenase （DHA）, substrate-induced DHA and alkaline phosphatase activities revealed the oligotrophic nature of the basal portion （320-480 cm）. Abundant bacterial isolates obtained from different depths were found to be clustered in 12 different phylogenetic groups by amplified ribosomal DNA restriction analysis. 16S rRNA gene sequencing revealed the dominant bacterial ribotypes to be affiliated to Firmicutes （Families Bacillaceae and Staphyloeoccaeeae） and Aetinobaeteria （Family Brevibaeteriaceae） with minor contribution of Proteobacteria （Families Phyllobacteriaeeae and Bartonellaceae）, pointing their endurance and adaptability to environmental stresses. Statistical analysis indicated that sediment organic carbon, salinity, total available nitrogen and total available phosphorous are most likely critical determinants of microbial activity in the Khadir Island sediments.     

Heterotrophic Production of Omega-3 Long-Chain Polyunsaturated Fatty Acids by Trophically Converted Marine Diatom Phaeodactylum tricornutum

We have created via metabolic engineering a heterotrophic strain of Phaeodactylum tricornutum that accumulates enhanced levels of the high value omega-3 long chain polyunsaturated fatty acid (LC-PUFAs) docosahexaenoic acid (DHA). This was achieved by generation of transgenic strains in which the Δ5-elongase from Ostreococcus tauri was co-expressed with a glucose transporter from the moss Physcomitrella patens. This double transformant has the capacity to grow in the dark in liquid medium supplemented with glucose and accumulate substantial levels of omega-3 LC-PUFAs. The effects of glucose concentrations on growth and LC-PUFA production of wild type and transformed strains cultivated in the light and dark were studied. The highest omega-3 LC-PUFAs accumulation was observed in cultures grown under mixotrophic conditions in the presence of 1% glucose (up to 32.2% of total fatty acids, TFA). Both DHA and EPA are detected at high levels in the neutral lipids of transgenic cells grown under phototrophic conditions, averaging 36.5% and 23.6% of TFA, respectively. This study demonstrates the potential for P. tricornutum to be developed as a viable commercial strain for both EPA and DHA production under mixo- and heterotrophic conditions.     

Factors regulating acetylene reduction assay for measuring heterotrophic nitrogen fixation in water-logged soils

In the C₂H₂-C₂H₄ assay for measurement of heterotrophic N₂ fixation in water-logged soils, the diffusion of C₂H₂ into the soil and the recovery of C₂H₄ from it are critical factors regulating the assay result. To establish an C₂H₂-C₂H₄ assay technique suitable for waterlogged soils, the C₂H₂-reducing activities (ARA), assayed by varying the method of assay gas filling, the pC₂H₂ of the assay gas, the duration of assay incubation and of soil vibration before the gas sampling, were compared.

A maximum ARA was measured when the following set of procedures were applied to the soil sample in assay flasks: 1) a 4-fold repetition of I-min evacuation under 0.01 atmospheric pressure and the subsequent I-min filling under 1 atmospheric pressure with assay gas at pC₂H₂ of 0.1 atm, 2) an assay incubation for 3 hr, and 3) a sampling of an aliquot of the headspace gas after strongly vibrating the flask for 1 min.

The ARA measured by this technique was several times larger than those measured by the techniques hitherto applied, and corresponded to an almost 80% of the V _max of the sample. This technique was, therefore, proposed for the assay of heterotrophic N₂ fixation in waterlogged soils.

A striking depression of ARA in the soil sample prepared with agitation indicated that a microbial ecosystem established in the soil should be kept as undisturbed as possible throughout the C₂H₂-C₂H₄ assay.     

人工培养牟氏角毛藻对弧菌抑制效果研究

研究了不同接种密度的牟氏角毛藻培育系统对2种弧菌的抑制效果及其相关性。结果表明牟氏角毛藻培育系统能够抑制2种弧菌的生长,并随着接种密度增加其抑制弧菌所需时间缩短;其中抑制副溶血弧菌的效果好于溶藻弧菌,2种弧菌均可利用牟氏角毛藻营养盐进行快速生长,但是它并没有促进或抑制角毛藻细胞的生长;加入弧菌的角毛藻培育系统中异养细菌的生长随着角毛藻的生长而变化,但是当角毛藻生长到后期,总的异养细菌均达到一个稳定值(约2×105CFU·mL-1),其中A号异养细菌远远超过其它异养细菌的量。A号异养细菌和角毛藻亲和力强而且能相互促进生长。     

一株异养硝化细菌的分离鉴定和脱氮特性研究

筛选对高浓度NH3-N养殖废水具有高效硝化能力的菌株,研究其硝化性能。通过比较几种已报道的筛选方法和不同生境中异养硝化细菌筛选效果,确定了以乙酰胺为唯一碳源和氮源,从高氨氮生境中可以筛选到高效的异养硝化细菌;进一步通过富集培养分离,从沼气池出水口水中分离到一株异养硝化细菌,并根据部分长度的16S rDNA序列进行了系统发育分析。该菌株具有高效异养硝化功能,在初始氨氮浓度为104 mg·L-1的异养氨化培养基中培养12 h后,氨氮和总氮去除率分别达81.7%和53.7%,最终氨氮和总氮去除率可达90.1%和61.3%,且培养液中无明显的硝酸盐氮和亚硝酸盐氮累积。16S rDNA的序列分析鉴定,该菌株与Paracoccus denitrificans具有99%相似性,结合生理生化分析认定该菌株是一株脱氮副球菌,命名为Paracoccus denitrificans FJAT-14899。筛选出的菌株Paracoccus denitrificans FJAT-14899对氨氮具有高效的去除率,显示了良好的应用前景。     

长期定位施肥下黑土呼吸的变化特征及其影响因素

阐明长期不同施肥下的土壤呼吸特征及其影响机制对黑土区固碳减排研究至关重要。该研究基于1990年开始的国家土壤肥力与肥料效益监测网站-吉林省公主岭市黑土监测基地,选取不施肥(CK)、单施氮磷钾肥(NPK)、无机肥配施低量有机肥(NPKM1)、1.5倍的无机肥配施低量有机肥(1.5(NPKM1))、无机肥配施高量有机肥(NPKM2)和无机肥配施秸秆(NPKS)6个处理,明确了长期不同施肥下土壤总呼吸和异养呼吸的季节变化特征,并分析了土壤温度、水分、微生物量碳氮、铵态氮、硝态氮与土壤呼吸和异养呼吸的关系。结果表明:长期有机无机肥配施可以显著提高土壤有机碳、全氮、土壤速效磷、有效钾的含量和土壤活性有机碳库组分含量(P0.05);与不施肥相比,长期有机无机肥配施和无机配施秸秆处理分别显著增加土壤呼吸及异养呼吸碳累积排放量56.32%~86.54%和70.01%~100.93%;根系呼吸对土壤呼吸的整体贡献为23.68%~34.30%;相关分析表明,土壤呼吸速率和异养呼吸速率与土壤温度极显著正相关(P0.01),与土壤含水率呈显著负相关(P0.01),土壤温度可以分别解释土壤呼吸和异养呼吸变化的42.79%和39.61%;土壤微生物量碳氮、土壤硝态氮均与土壤呼吸速率和异养呼吸速率极显著相关(P0.01),土壤微生物量碳氮、土壤硝态氮可以分别解释土壤呼吸和异养呼吸变化的78.42%和77.18%,58.33%和56.79%,59.29%和59.14%;土壤铵态氮虽然显著影响土壤呼吸速率(P0.05),可以解释土壤呼吸变化的5.56%,但其对异养呼吸速率的影响不显著。综合来看,微生物量碳对土壤呼吸及异养呼吸的影响最大,而土壤含水率(15%)越高则土壤呼吸越弱;无机配施秸秆处理可以提高土壤碳库组分含量,且作物生育期内土壤呼吸及异养呼吸碳累积释放量均低于等氮量下施用有机肥(NPKM1)的处理,为最佳的农田管理措施。     

草坪土壤异养硝化菌的分离筛选及硝化产物特征分析

从早熟禾草坪土壤中分离出两株异养硝化菌,将菌株16S rDNA测序结果在GenBank中进行同源性对比确定菌种,用无机氮源（NH4+-N和NO3--N）在适宜条件下测定两个菌株的硝化能力,分析中间产物、反硝化作用和N2O排放特征。结果表明,分离到的异养硝化菌属于假单胞杆菌属（Pseudomonas sp.）,两个菌株的16S rDNA片段长度分别为1 448和1 460 bp,命名为Pseudomonas sp. BJ和Pseudomonas sp. DJ,GenBank登录号分别为MF001078和MF001079;两个菌株对无机氮的氧化能力很强,培养24 h内BJ和DJ菌株对铵态氮的总硝化率分别为96.61%和97.06%,最大硝化速率分别为21.97和18.90 mg ? L-1 ? h-1。但两个菌株的反硝化能力不强,最大反硝化速率分别为0.99和1.00 mg ? L-1 ? h-1;两个菌株的代谢途径是NH4+ → NH2OH → NO2- →NO3-,DJ菌株的最终产物以NO3--N为主,积累量高于BJ菌株（P < 0.05）。BJ菌株的N2O排放量显著高于DJ菌株（P < 0.05）。