潮间带沉积物厌氧烃降解细菌的多样性及Desulfovibrio subterraneus ND17的分离鉴定

潮间带作为海陆交界处,易受到来自海洋的石油污染,且各类石油烃进入沉积物后的降解过程尚不清楚。前人在各类生境中对好氧微生物烃降解方面已有较多研究,但对近海潮间带环境中的厌氧烃降解鲜有报道。本研究对青岛女岛湾潮间带沉积物深层样品以混合烃（中长链烷烃、多环芳烃）为碳源,硫酸盐作为电子受体进行厌氧富集培养。富集菌群的细菌多样性表明在混合烃作为碳源的作用下,优势菌群转变为脱硫叠球菌科（Desulfosarcinaceae）、脱硫杆菌科（Desulfobacteraceae）等具有石油烃降解潜力的硫酸盐还原菌。经分离纯化得到一株厌氧烃降解菌ND17,与地下脱硫弧菌属模式种Desulfovibrio subterraneus HN2T 16S rRNA基因序列的相似度为99.93%。进一步实验表明,菌株ND17在厌氧条件下对二十四烷和菲的降解率可分别达到53.9%和35.7%。这也是首次对脱硫弧菌属单菌在厌氧条件下进行石油烃降解的研究。脱硫弧菌作为一种广泛分布在厌氧环境的细菌,本研究为进一步认识其在海洋石油污染环境中的修复潜力提供了支撑。     

山东省渤海海洋保护区典型海域表层海水微生物群落多样性分析

海洋微生物在生态系统循环中发挥重要作用。为系统了解山东省渤海海洋保护区表层海水微生物群落多样性,作者利用Illumina高通量测序及生物信息学分析技术研究了12个水体样本的菌群构成和多样性特征。实验结果表明,山东渤海海洋保护区典型海域表层海水微生物群落丰富,12个水体样品OTU分属于25门、56纲、103目、163科、227属;门、纲、目、科、属分类等级上的优势类群分别为变形菌门(Proteobacteria)(57.95%)、α-变形菌纲(Alphaproteobacteria)(21.88%)、黄杆菌目(Flavobacteriales)(4.31%)、红细菌科(Rhodobacteraceae)(10.68%)、Candidatus portiera(7.94%);保护区内表层海水微生物具有其独特的菌群特征和多样性。本研究结果为进一步认识海洋微生物多样性与海洋生态环境保护的关系奠定了基础。     

长江口表层沉积物中可培养有机解磷菌多样性特征

为研究不同水文条件下长江口邻近海域表层沉积物中可培养解磷细菌的多样性,选择此海域20个站位,针对2017-02(枯水期)、2017-05(平水期)和2017-07(丰水期)取得的表层沉积物,利用卵磷脂选择性培养基筛选解磷菌。结果表明,从长江口表层沉积物中筛选获得的572株可培养有机解磷菌,分属于Firmicutes,Proteobacteria和Actinobacteria中19个属80个种。其中,从2017-02样品获得136株好氧解磷菌,优势属为嗜冷杆菌属(Psychrobacter),占比69.85%;从2017-05样品中获得123株好氧解磷菌,12株兼性厌氧解磷菌,优势属为芽孢杆菌属(Bacillus),占比54.81%;从2017-07样品中获得100株好氧解磷菌,201株兼性厌氧解磷菌,优势属为弧菌属(Vibrio),占比87.71%。同时平板解磷能力相对较强的菌株也主要为优势种属Psychrobacter和Vibrio。通过Shannon和Simpson多样性指数分析,发现解磷菌多样性程度为春季冬季夏季。实验结果表明,长江口表层沉积物中可培养有机解磷菌多样性丰富,同一区域内沉积物中的有机解磷菌群落结构存在季节差异。长江口表层沉积物中丰富的有机解磷细菌的存在必将是磷循环重要的参与者,可能在区域物质循环和环境问题中发挥了重要的作用。     

白令海和楚科奇海表层沉积物中多环芳烃降解微生物多样性

为了研究白令海(海盆及陆架)至楚科奇海陆架表层沉积物中多环芳烃(PAHs)降解菌的多样性,并获得新的PAHs降解菌资源。在GC-MS分析沉积物中PAHs种类和含量的基础上,以萘、菲和芘的混合物为唯一碳源和能源对表层沉积物样品进行富集,通过平板分离鉴定可培养菌株,并验证其降解能力;同时利用变性梯度凝胶电泳(DGGE)和Illumina高通量测序技术分析降解菌群结构。GC-MS测定结果表明,14个表层沉积物中PAHs总干质量介于32.99~276.97ng/g。富集菌群中共分离获得51株可培养细菌,平板纯培养、PCR-DGGE及Illumina测序结果均表明,菌群中优势的降解菌是γ-proteobacteria的Marinobacter,Pseudoalteromonas,Pseudomonas和Actinobacteria门的Dietzia菌。此外,Illumina测序结果还表明14个降解菌群在菌群结构组成上,可分为海盆区和陆架区两种类群;同时检测到一些低丰度的海洋专属PAHs降解菌,如Cycloclasticus,Alteromonas和Neptunomonas等。本文结果将加深对白令海及楚科奇海表层沉积物中PAHs降解菌资源与生物多样性的认识。     

应用高通量测序技术分析北方刺参养殖池塘环境菌群结构

刺参(Apostichopus japonicus)是一种经济价值很高的海珍品,具有很高的药用价值和营养价值,本文采用高通量测序技术分析了大连地区3个养殖公司刺参养殖环境中室内养殖池塘水体、室外养殖水体和沉积物中菌群结构特征,9个样品所得有效序列为26503—37825条,可归为1502—5741个分类操作单元(OTUs)。研究结果表明:(1)刺参养殖环境中具有较高的菌群多样性,其中3个公司的刺参养殖沉积物环境中菌群多样性较其相应公司刺参养殖水环境中菌群多样性高;(2)9个样品中的细菌可归为26个门类,除常见报道的变形菌门、拟杆菌门、绿弯菌门、浮霉菌门、蓝细菌门、酸杆菌门、疣微菌门、厚壁菌门和放线菌门外,还有17个门类的鲜见报道的菌群被检测到;(3)各样品中菌群组成虽有差异,但变形菌门和拟杆菌门为优势菌,约占50.54%—99.91%。     

高通量测序分析涠洲油气田开发区的微生物群落结构

基于16S r RNA基因,采用Illumina Mi Seq高通量测序技术分析和比较了涠洲油气田开发区表层海水和沉积物中微生物群落结构。Shannon多样性指数表明涠洲油气田微生物多样性较高,并且表层海水的微生物多样性要高于沉积物。表层海水中微生物以芽孢杆菌Bacilli居多,其次为蓝细菌Cyanobacteria、α变形菌Alphaproteobacteria和γ变形菌Gammaproteobacteria;而沉积物中微生物群落主要类群为γ变形菌。维恩图表明涠洲油气田表层海水的微生物组成或沉积物的微生物组成各自具有一定的相似性,热图和主成分分析则发现该两类环境介质之间的微生物结构差异较大。Illumina Mi Seq高通量测序还发现涠洲油气田存在潜在石油类降解菌假单胞菌属Pseudomonas、鞘氨醇单胞菌属Sphingomonas、红球菌属Rhodococcus和分枝杆菌属Mycobacterium,并且沉积物中该四类微生物相对比例要高于表层海水,这一现象可能由环境中石油类含量决定。研究将为评价油气开发活动对微生物群落的影响提供依据,并为海洋环境中石油污染的生物修复提供基础数据。     

辽河口沉积物中古菌和细菌群落结构分析

本研究旨在了解辽河口表层沉积物中古菌和细菌的群落结构组成、多样性及其与环境因子之间的相关性。采用构建古菌和细菌16S rRNA基因文库的方法，应用Illumina Miseq测序技术进行序列分析。结果表明:辽河口表层沉积物中细菌多样性高于古菌多样性，近岸细菌和古菌多样性高于远岸，即河相区（0.8~7.04）细菌多样性高于混合区（13.1~20.7）和海相区（24.2~31.5）；主要古菌群落为奇古菌门（Thaumarchaeota，72.73%）和广古菌门（Euryarchaeota，25.05%），其中泉古菌门（Crenarchaeota，0.001%）只在河相区站位被发现；细菌群落组成中变形菌门（Proteobacteria，61.94%）为该河口的优势菌群，其次为拟杆菌门（Bacteroidetes，11.21%）和酸杆菌门（Acidobacteria，5.59%），其他门类如蓝藻门（Cyanobacteria，3.03%）等比例较小。与环境因子的冗余度分析表明:影响表层沉积物中古菌群落分布的主要环境因子依次为氨盐、泥、酸碱度、盐度、电导率和砂，而细菌群落的分布主要受到溶氧、泥、砂、黏土和总磷的影响。由此可见，不同环境条件下微生物的群落结构存在空间异质性，不同微生物对同一环境条件的响应亦不同。     

南沙深海沉积物中石油降解菌的分离鉴定和多样性分析

为了解我国南沙海域中石油降解菌的多样性,认识其在环境自净中作用并获得用于石油污染生物修复的菌种资源,以不同链长烷烃及支链烷烃对南沙深海沉积物样品进行富集与烃降解菌的分离鉴定,并结合16S rRNA基因文库和DGGE技术对降解菌群的结构进行了分析.结果表明：通过16S rRNA基因文库构建及PCR-DGGE分析发现,不同链长烷烃富集后的菌群中,食烷菌（Alcanivorax）在各降解菌群中都是绝对优势菌;另外,除烃海杆菌（Marinobacter hydrocarbonoclasticus）、海绵假单胞菌（Pseudomonas pachastrellae）,以及Idiomarina与Kangiella属的细菌也是菌群中较重要的优势菌.此外,通过2种平板培养基从烷烃富集的降解菌群中分离获得了8个不同属的细菌.本研究还首次发现Kangiella属（食烷菌科）的细菌可能参与了烷烃降解.南沙沉积物烷烃降解菌多为γ-Proteobacteria;其中,食烷菌科（Alcanivoraceae）（Alcanivorax与Kangiella属）的细菌在降解菌群中是绝对优势菌,假单胞菌与海杆菌在烷烃降解过程中也起着重要作用.     

深海沉积物中龙须菜降解菌群的筛选与鉴定及多样性分析

以龙须菜为唯一的营养源对深海沉积物进行富集和筛选,获得一个可降解龙须菜(Gracilaria lemaneiformis)、紫菜(Porphyra umbilicalis)和海带(Laminaria japonica)产生还原性寡糖的菌群.通过16S rRNA序列及16S rRNA-RFLP分析对该菌群中的细菌多样性进行了研究.结果表明,降解菌群中的细菌组成主要为弧菌属(Vibrio,8株)、火色杆菌属(Flammeovirga,7株)和希瓦氏菌属(Shewanella,5株),其中希瓦氏菌属和火色杆菌属的细菌在菌群中的丰度较高.采用龙须菜为唯一营养源的筛选培养基对菌群中的细菌进行分离,获得4株具有琼胶酶活力的细菌,包括2株火色杆菌属和2株希瓦氏菌属细菌.培养和未培养的结果均表明火色杆菌和希瓦氏菌这2个属为所研究的深海沉积物主要的龙须菜降解菌.对原始降解菌群和所分离的关键菌株进行了龙须菜酶解产糖能力的比较,结果发现菌群中的弧菌属菌株虽然自身没有酶解龙须菜的能力,但可能可以协助关键菌株,提高菌群对龙须菜的降解效率.因此本研究中所获得的菌群和菌株有望在琼胶寡糖的绿色生产中得到广泛应用.     

南海深海氮循环微生物的原位培养与多样性分析

借助自主研发的深海水体原位定植培养系统,在南海3 300 m的深海水体中进行了氮循环微生物的原位培养.通过向富集仓中投加缓释肥(铵盐、硝酸盐和尿素)进行了为期17个月的原位富集.结合高通量测序技术和分离培养的方法,对深海原位富集物及实验室二次富集物进行了微生物多样性分析. 16S rRNA基因高通量测序结果表明,在南海原位富集样品中,细菌以变形菌门丰度最高,富集仓内脱脂棉附着介质和仓内水样中的最优势属分别为希瓦氏菌属(Shewanella)和科韦尔氏菌属(Colwellia);古菌以奇古菌门为主,其中氨氧化古菌(AOA)所占比例很高.通过平板培养分离到17株细菌,主要包括盐单胞菌属(Halomonas)、海杆菌属(Marinobacter)和亚硫酸盐杆菌属(Sulfitobacter)等.通过不同起始氮源(氨氮、硝酸盐、亚硝酸盐)和培养温度(28℃和10℃),对原位富集样品进行实验室二次富集,获得了5个富集菌群.分析发现,它们均具有较好的脱氮效果,可以产生N_2O或N_2;这些菌群均以盐单胞菌属占绝对优势,其次为海源菌属(Idiomarina)或海杆菌属,其中盐单胞菌和海源菌成功获得分离.单菌功能验证表明,分离获得的盐单胞菌具有好氧反硝化能力,其在原位环境下反硝化活性尚有待确认.     

南、北极近岸海水中好氧不产氧光合基因pufM的多样性

好氧不产氧光合(AAP)细菌因其具有利用溶解性有机物及光能的能力,在海洋碳循环及能量流动中发挥着重要的作用。该类细菌广泛分布在海洋环境中,其在不同生境中的多样性已被调查。但到目前为止,人们对于高纬度地区好氧不产氧光合细菌的认识还较为缺乏。有鉴于此,本研究基于编码光反应复合物上一个色素结合蛋白亚基的pufM基因,对北极王湾及南极乔治王岛近岸水体中夏季好氧不产氧光合细菌的多样性进行了检测。针对2个王湾站位和2个南极麦克斯维尔湾站位构建了4个pufM基因克隆文库,获得674个阳性克隆子。北极克隆子全部由α-变形细菌组成,而南极克隆子则包括α-变形细菌及β-变形细菌。来源于类似红细菌科的pufM基因在所有样品中皆占据优势。此外,与一株滴状亚硫酸盐杆菌中质粒编码的pufM基因存在亲缘关系的序列,在南、北极样品中均占优势。结果表明海洋环境中的pufM基因存在跨极甚至是环球分布。与此同时,南、北极序列之间的差异,也表明了极地地方种的存在。这些结果显示,作为好氧不产氧光合细菌的红细菌科在两极的近岸水体中具有重要的地位。     

日本真海带对硒的积累和生物转化

在海水中添加亚硒酸盐(Na₂SeO₃)及不同浓度的氮磷营养盐(NaNO₃+NaHO₂PO₄),研究海带对硒的积累,通过分离测定海带富硒前后各生化组分的含硒量来探讨硒的生物转化问题.结果表明天然海带含硒量(鲜重)为0.451~0.596μg/g,海带富硒最佳的Na2SeO₃浓度为200mg/dm3,培养56h后的富硒倍率约为50.在天然海带中,硒主要以有机态存在,有机硒占总硒含量的86.22%;在含有200mg/dm3NaO₂SeO₃的海水中培养56h后测得海带的总硒为24.481μg/g,其中有机硒含量为16.703μg/g,无机硒为4.714μg/g;在添加氮磷营养盐的含硒海水中,海带的富硒能力得到进一步的提高,在适宜的氮磷条件下(150mg/dm3NaNO₃和25mg/dm3NaHO₂PO₄)总硒含量达到33.649μg/g,而无机硒含量基本不变,为4.497μg/g,因此提高部分转化为有机硒,有机硒含量达到24.678μg/g.这说明海带具有通过自身代谢将无机硒转化为有机硒的较强的能力.     

Characterization of the proteinaceous matter in marine aerosols

Marine aerosols play a dominant role in the transfer of oceanic material to the atmosphere. Most marine aerosol originates when air bubbles burst at the sea surface ejecting material from the sea surface microlayer and bubble surface layers into the air. Concentrations of chemical compounds in these surface layers often differ from their concentrations in bulk water. We examined the enrichment of aerosols with proteinaceous matter and attempted to characterize the physical nature and sources of this matter. We measured concentrations of dissolved free (DFAA), dissolved combined (DCAA), and particulate (PAA) amino acids, transparent stainable particles (TSP), and bacteria and virus-like particles as carriers of protein, in natural and simulated aerosols. We also evaluated D/L ratios certain amino acids in all amino acid fractions.DFAA and DCAA enriched the aerosols we sampled by 1.2–20 times compared to bulk seawater; PAA enrichment was usually higher (up to 50-fold). Aerosols contained particles typical of seawater, e.g., microorganisms, organic debris, inorganic particles with adsorbed organic matter, but also a large number of semitransparent gel-like particles, which all contained amino acids. Some of these particles were probably scavenged from bulk water, but new particles produced as bubbles burst at the surface comprised at least 10% of total proteinaceous matter in the aerosol. D/L ratios of certain amino acid suggested that the particles were most likely made from dissolved polymers secreted by phytoplankton that were concentrated on bubble surfaces and in the microlayer. Examination with Alcian Blue (a dye that targets carbohydrates) and Coomassie Blue (a dye that targets proteins) showed that most TSP in the aerosols contained both proteins and polysaccharides. Microorganisms enriched the aerosols by up to two orders of magnitude, but contributed less than 4% to the total protein pool.     

A study of crude oil‐degrading bacteria from mangrove forests in the Persian Gulf

Mangroves are coastal ecosystems, found in tropical and subtropical regions around the world. They are found in the transitional zones between land, sea, and rivers. Petroleum hydrocarbons are the most common environmental pollutants, and oil spills pose a great hazard to mangroves forests. This research was focused on the isolation and characterization of crude oil‐degrading bacteria from mangrove ecosystems at the Persian Gulf. Sixty‐one crude oil‐degrading bacteria were isolated from mangrove samples (plant, sediment, and seawater) that enriched in ONR7a medium with crude oil as only carbon source. Some screening tests such as growth at high concentration of crude oil, bioemulsifier production, and surface hydrophobicity were done to select the most efficient strains for crude oil degradation. Molecular identification of strains was carried out by amplification of the 16S rRNA gene by PCR. The results of this study were indicated that the quantity of crude oil‐degrading bacteria was higher in the root of mangrove plants compare to other mangrove samples (sediment and seawater). Also, identification results confirmed that these isolated strains belong to Vibrio sp. strain NW4, Idiomarina sp. strain BW32, Kangiella sp. strain DP40, Marinobacter sp. strain DW44, Halomonas sp. strain BS53, and Vibrio sp. strain DS35. The application of bioremediation strategies with these bacteria can reduce crude oil pollution in this important marine environment.     

Microbial diversity in polluted harbor sediments II: Sulfate-reducing bacterial community assessment using terminal restriction fragment length polymorphism and clone library of dsrAB gene

Sulfate-reducing bacteria (SRB) are important regulators of a variety of processes in coastal marine sediments regarding organic matter turnover, biodegradation of pollutants, and sulfur and carbon cycles. Yet their community compositions have not been investigated in polluted harbor sediments. This study described the diversity and spatial variation of SRB communities in surface sediments in Victoria Harbor, Hong Kong. The spatial variation of SRB communities was described by terminal restriction fragment length polymorphism (T-RFLP). The results showed that the most diversified terminal restriction fragments were found at polluted sites. In addition, cluster analysis indicated that although the SRB communities were different at the two polluted sites, they were still more similar to each other than to the two more distant reference sites. Based on a dsrAB clone library constructed at a polluted site, diversified SRB were found, represented by 30 Operational Taxonomic Units (OTUs). Upon comparisons among the SRB sequences detected from this study and those in the GenBank, five clades of SRB were found. Three clades belonged to the known families Desulfobacteraceae, Desulfobulbaceae, and Syntrophobacteriaceae. The majority of sequenced clones, which distantly related to sequences in the GenBank, constituted the remaining two unclassified groups, suggesting unique SRB members related to the polluted harbor environment. Statistical analyses indicated that estimated SRB richness correlated with environment factors such as sulfur content, acid volatile sulfate, and redox potential.     

Quantitative Benthic Community Structure in Blanca Bay and Its Relationship With Organic Enrichment*

Abstract. A two year benthic sampling program of a 2000 km² area, covering 22 stations, showed a dominance of deposit-feeding polychaetes. The quantitative structure of the three infaunal assemblages was related to a salinity gradient: the inner and outer regions were inhabited by, respectively, mixohaline and euhaline associations, both quantitatively related to granulometric facies of sediment. Species diversity was very low in the inner region, although abundance of opportunistic species was high. In the outer region, abundance and diversity were more stable, with an increasing number of species. A comparison of species number and abundance with Species-Abundance-Biomass curves from enriched environments showed that the inner region of Bianca Bay may be considered as an organically enriched environment. Scoloplos, a species associated with organic enrichment, and dense terebellid belts were present in great abundances in front of sewage outflows. The spatial distribution of heterotrophic bacteria and the bivalves Corbula and Nucula also followed this pattern.     

Vertical and seasonal differences in biogenic silica dissolution in natural seawater in Suruga Bay, Japan: Effects of temperature and organic matter

Vertical and seasonal characteristics of biogenic silica (BSi) dissolution in seawater were investigated by multiple dissolution experiments using seawater collected from surface and mesopelagic layers in Suruga Bay during the period 2002–2004. The dissolution rate coefficients calculated based on temporal changes of BSi concentration varied with the season of sample collection. They ranged from 0.023–0.057 day^− 1 for surface samples and 0.0018–0.0025 day^− 1 for mesopelagic samples for temperatures approaching in situ conditions. Experiments at various temperatures confirmed that BSi dissolution depends on temperature in natural seawater. Dissolution rate coefficient (day^− 1) of BSi correlated significantly with temperature (°C), and Q₁₀ was 2.6. Addition of bioavailable organic matter to low-bioactivity seawater enhanced the protease activity and abundance of bacteria, and increased BSi dissolution rate by a factor of 1.4–2.0. There is clear evidence that BSi dissolution is accelerated by bacterial activity and potentially limited by bioavailable organic matter in natural seawater. Dissolution rates and total decreases of BSi concentration were lower during experiments using mesopelagic samples than in those using surface samples. This suggests that dissolution of BSi varies with depth and that BSi in the mesopelagic water is more resistant to the dissolution than that in the surface water. This lower dissolution rate was caused by lower temperature and lower bacterial activity due to less bioavailable organic matter in mesopelagic water. Our results provide a mechanistic understanding of variations in silica cycling within the seasonally and vertically differing marine environment.     

Photochemical production of low-molecular-weight carbonyl compounds in seawater and surface microlayer and their air-sea exchange

Coastal and oceanic surface microlayer samples were collected using a stainless steel screen, along with subsurface bulk seawater, and were analyzed for low-molecular-weight (LMW) carbonyl compounds, including formaldehyde, acetaldehyde, propanal, glyoxal, methylglyoxal, glyoxylic acid and pyruvic acid. The enrichment factor in surface microlayer compared to corresponding subsurface seawater ranged from 1.2 to 21. A time-series measurement at a coastal site showed strong diurnal variations in concentrations of the LMW carbonyl compounds in the surface microlayer and in the enrichment factor, with maxima in the early afternoon and minima in the early morning. Exposure of samples to sunlight resulted in the higher yields of these compounds in the surface microlayer than in the bulk seawater, by a factor of 1.1–25, suggesting that the higher photoproduction rate of LMW carbonyl compounds in the surface microlayer accounts for the majority of the observed enrichment in these samples. Potential sinks include biological uptake and mixing. Air-sea exchange may be a source for soluble compounds and a sink for less soluble compounds. The enrichment of the LMW carbonyl in surface microlayer may alter their net air-sea exchange direction e.g., from the ocean as a potential sink to a source for atmospheric acetaldehyde and acetone. The residence times of the LMW carbonyl compounds in the microlayer were estimated to be on the order of tens of seconds to minutes using a modified two-layer model. However, to maintain the observed microlayer enrichment factor, the residence time should be on the order of ˜ 1 hour. This prolonged residence time may be due to organic enrichment in the surface microlayer (‘organic film’) which inhibited molecular transfer of carbonyl compounds into and out of the microlayer. The deviated behavior from model prediction may also be due to changes in the apparent partition coefficients of these species as a result of thier physical and chemical interactions with organic matrix in the surface microlayer.     

Microbiological Studies Relevant to a Lobster Introduction into Fatty Basin,British Columbia

Studies in marine microbiology relevant to the cultivation of lobster in Fatty Basin were made. Biomass of bacteria and allied microorganisms in whole seawater column of the basin was very small (2×10⁴ gC), but a large biomass was found in the sediments (3×10⁵ gC). The decomposition of chitin occurred chiefly in the sediments. The rate of decomposition (500 g/day) was approximately half of the rate of production. However, the remaining production was considered not to be involved in the chitin cycle of the basin. This hypothesis was supported by the results of the analysis of the budget of organic matter in the area. Shell disease of lobster caused by chitinoclastic bacteria was detected, although it was not serious. A destructive yeast parasite of crustaceans,Metschnikowia, was collected only from a crab in the basin. This report is dedicated to Professor KenSugawara for his 70 years old anniversary.