张瑞蕊1, 薛梅2, 李宁宁1, 靳亚梅2, 魏小晶2, 倪永清2
1.石河子大学生命科学学院, 新疆 石河子 832000;
2.石河子大学食品学院, 新疆 石河子 832000
收稿日期：2018-04-12；修回日期：2018-08-18；网络出版日期：2018-11-29
基金项目：国家自然科学基金（40961002，41140009，41271268）
*通信作者：倪永清, Tel:+86-993-2058095;E-mail:niyqlzu@sina.com
摘要：[目的] 揭示乌鲁木齐河源天山1号冰川表面冰尘（CS）和底部沉积层（DS）可培养酵母菌系统发育类群及其结构组成差异，分析低温酵母菌代表菌株之间的生态、生理生化特性。[方法] 利用4种培养基分离天山1号冰川可培养酵母菌，采用ITS基因序列分析确定菌种的系统进化地位。对分离菌株的最适生长温度、耐盐性和产酶等生态、生理学特性进行分析。[结果] 从冰尘和底部沉积层中共分离出152株酵母菌菌株，通过ITS rRNA基因序列的NCBI比对和Rep-PCR指纹分型，结果表明酵母菌类群包括担子菌门（Basidiomycota）和子囊菌（Ascomycota），分属于14个属26种，其中担子菌门柄锈菌亚门（Pucciniomycotina）88株、伞菌亚门（Agariomycotina）24株，子囊菌门40株，冰川广布酵母菌Vishniacozyma victoriae为优势菌株（占比21.84%）。17种酵母的最适生长温度为15 ℃、2种为10 ℃、6种为20 ℃。25株代表酵母菌株产酶分析显示，产脂肪酶、淀粉酶、蛋白酶菌株分别为11株、11株、5株，6株3种酶都不产。[结论] 天山1号冰川冰尘及底部沉积层可培养低温酵母系统发育类群结构存在差异，产低温酶活性高、稳定性好，为今后冰川低温酵母菌的研究提供有价值的数据支持。
关键词：天山冰川冰尘底部沉积层可培养酵母多样性产酶活性
Phylogeny and diversity of culturable yeasts in supraglacial cryoconite and subglacial sediments of the Glacier No.1 in Tianshan Mountains
Ruirui Zhang1, Mei Xue2, Ningning Li1, Yamei Jin2, Xiaojing Wei2, Yongqing Ni2
1.School of Life Sciences, Shihezi University, Shihezi 832000, Xinjiang Uygur Autonomous Region, China;
2.School of Food Sciences, Shihezi University, Shihezi 832000, Xinjiang Uygur Autonomous Region, China
Received 12 April 2018; Revised 18 August 2018; Published online 29 November 2018
*Corresponding author: Yongqing Ni, Tel: +86-993-2058095; E-mail: niyqlzu@sina.com
Supported by the National Natural Science Foundation of China (40961002, 41140009, 41271268)
Abstract: [Objective] The objective of this study was to elucidate the phylogenetic group and diversity of structure composition of culturable yeasts isolated from supraglacial cryoconites and subglacial sediments of the Glacier No.1 in Tianshan Mountains and to analyze the physiological and biochemical characteristics of different low temperature yeasts. [Methods] Culturable yeasts were detected on rose Bengal agar (RB), clichloran 18% glycerol agar (DG18), malt extract yeast extract soytone agar (MYP) and yeast-malt extract agar (YMP) media. The phylogenetic position of yeasts was determined by internal transcribed spacer (ITS) gene sequence. The physiological and biochemical tests included optimal growth temperature, salinity tolerance and enzymatic activities. [Results] A total of 152 culturable yeasts were isolated from supraglacial cryoconites and subglacial sediments. The results of National Center of Biotechnology Information (NCBI) alignment of internal transcribed spacer (ITS) rRNA gene sequence and Rep-PCR fingerprinting showed that the yeast groups included Basidiomycota and Ascomycota, belonging to 14 genera and 26 species. Among them, 88 strains were Pucciniomycotina, 24 strains were Agariomycotina, and 40 strains were Ascomycota. Vishniacozyma victoriae was the dominant strain (21.84%). The optimum growth temperature for 17 yeasts was at 15 ℃, 2 strains at 10 ℃ and 6 strains at 20 ℃. The analysis of lipase, protease and amylase activity showed that 11 of the 25 low temperature yeasts produced lipase and 11 produced amylase and 5 produced protease, 6 strains did not produce any enzymes. [Conclusion] The results of this study provide valuable data for the research of community structure and physiological characteristics of cryogenic yeasts in glaciers.
Keywords: glaciers in the Tianshan Mountainscryocnitesubglacial sedimentsculturable yeastsdiversityenzyme activity
地球上冰川等冰冻圈极端环境大约占地球表面积的10%，包括南极、北极、西伯利亚等环北极冻土以及中低纬度高山、高原冰川、冻土等[1]。生存于冰冻圈环境的微生物由于长期适应低氧、寡营养、强辐射、干燥、冻融等极端环境，进化出独特的生理生态学特征，蕴含了独特的微生物技术潜力和基因库资源[2]。真菌不仅物种多样性非常丰富，其生存适应能力比细菌更强，在自然界分布也更广泛。酵母菌作为真菌一大类群，广泛存在于土壤、岩石、沉积物、海洋、河流、湖泊等生境[3]，在极端的冰冻圈环境也广泛分布，不仅对低温环境中的生物地球化学循环发挥重要作用，在工农业生产、医药等生物技术领域和污染生境治理等方面都有潜在的开发价值[4]。
研究发现，冰川冰芯[5]、底部沉积层[6]、周边冻土层[7]、冰川积雪[8]及冰川岩石[9]等环境中生存着不同的功能微生物类群。来自陆地的灰尘、海洋表面气溶胶和火山灰烬等携带外源微生物粘附在积雪、矿物质或者有机质颗粒上沉降在冰川表面，部分微生物代谢产生胞外多糖，通过粘附作用在冰川表面形成深色的堆状冰尘(cryocionite)[10]。冰尘吸收太阳辐射能量后在冰川表面形成冰尘空洞，并积累丰富的有机质，使得各类微生物得以生存。相比之下，冰川底部由冰、水、岩沉积层构成，为缺氧、缺光照、高静压、持续低温(0 ℃之下)极端严酷的生态系统，是一个原始的、较少受到外源环境影响的寡营养生态环境。冰川酵母菌不仅可以在低温下生存、繁殖，甚至在零摄氏度以下也能维持其代谢活性[11]。但由于气候环境因素以及冰川融化形成径流的联通作用，冰川生态系统在空间上既存在联系，又呈现出明显的生境异质性[12]，不同的生境条件对低温酵母菌种群具有生态选择作用，共同决定了生境中酵母菌种群的组成。
研究发现，低温酵母菌具有特殊的功能，如能够产低温酶、合成活性大分子物质以及生物降解等，目前在生物、医药、洗涤、食品加工、环保等行业有极好的应用前景[13]。乌鲁木齐河源天山1号冰川是我国目前在相关科学研究领域数据最为详尽的冰川，尤其在冰川物质平衡、水文化学、退缩、径流和降水、气候变化、生态状况以及生物多样性等方面积累了丰富的研究成果[14-15]。目前对1号冰川不同生境中的微生物多样性已经有报道[16]，我们前期对冰川酵母菌已作了初步报道[17]，本文对天山1号冰川生态特征截然不同的表层冰尘和底部沉积层中可培养酵母菌的系统发育类群及生态生理特性进行了比较分析，目标是揭示在冰川局域范围内，生境对微生物多样性的影响以及生态选择作用，为冰川环境中潜在利用价值低温酵母菌资源的开发利用提供参考依据。
1 材料和方法 1.1 材料 样品采集于天山一号冰川(海拔3750 m，43°07.125N，86°48.707E)，2017年8月在冰川表面及冰川尾部取样，其中在冰川表面3个取样点，每个样点平行取样4份，样方长约1.5 m×1.5 m，每个样品取5份；冰川底部取样退缩后裸露的冰碶沉淀物。迅速将冰尘颗粒和处理过的沉积物装入己灭菌的保鲜盒内，置于冰箱中-4 ℃保存，8 h之内运回实验室，在24 h内取样处理分菌，并对样品理化性质进行测定。以上所有过程均在无菌条件下完成。
1.2 培养基
1.2.1 分离培养基: (1) RB培养基(g/L)：蛋白胨10.000，葡萄糖20.000，KH2PO4 1.000，MgSO4·7H2O 0.500，琼脂粉16.000，Rose Bengal 0.030，氯硝胺0.002，pH 6.5；(2) DG18培养基(g/L)：甘油18.000，蛋白胨5.000，葡萄糖10.000，KH2PO4 1.000，MgSO4·7 H2O 0.500，琼脂16.000，氯硝胺0.002，pH 5.6；(3) MYP培养基(g/L)：麦芽提取物7.0，酵母提取物0.5，大豆蛋白胨2.5，琼脂15.0，Triton X-100 0.2，pH 6.5；(4) YMP培养基(g/L)：麦芽提取物7，酵母提取物5，蛋白胨5，琼脂粉16，pH 6.5[18]。
1.2.2 基础培养基: YEPG培养基(g/L)：酵母提取物10，蛋白胨10，葡萄糖20，琼脂16，pH 6.5。
1.3 主要仪器和试剂 高速冷冻离心机为德国Thermo公司Fresco21型；光学显微镜为德国生产Leica DM3000；PCR仪为德国Biometra公司Tprofessional；凝胶成像系统为法国Vilber多功能成像系统；水平电泳仪为美国BioRad公司PowerPac Universal、电泳槽SUBCEILGT (20 cm×25 cm)；超微量分光光度计为德国Thermo Scientific Nano Drop 2000/2000C。DNA分子量marker及其他PCR扩增所需试剂均购自天根生化科技(北京)有限公司；其余试剂均为进口或国产分析纯；PCR引物购自上海捷瑞生物工程有限公司。
1.4 样品理化性质测定 测定天山一号冰川表面冰尘(CS)和底部沉积层(DS)样品中水分含量(SM)、pH值(水：冰尘= 2.5：1)、速效磷(AP)、速效钾(AK)、硝态氮(NO3--N)、有机质(OM)及全氮(TN)、全钾(TK)、全磷(TP)的总量[19]，每个样品设置3个平行，用SPSS 19软件进行统计学分析。
1.5 菌株的分离纯化 取10 g样品加入90 mL 0.9%生理盐水梯度稀释，分别吸取不同浓度梯度的100 μL已稀释样品溶液，合适梯度稀释液涂布于4种分离培养基，一式两份于15 ℃恒温培养5-7 d，待菌落长出，根据菌落颜色、大小、形态等表型差异进行初步分离，转接划线，将纯化的培养物转接到YEPD液体培养基上，4 ℃恒温保藏。
1.6 DNA提取 将已纯化的菌株转接到5 mL的YEPG液体培养基中，15 ℃培养扩大培养5-7 d，吸取1.5-2.0 mL培养的酵母菌液，5000 r/min离心5 min，弃上清，收集酵母菌细胞沉淀；参照Bergamo等[20]方法提取DNA，DNA提取后加入100 μL 1×TE溶解，-20 ℃保存。采用超微量分光光度计定量核酸浓度和纯度。
1.7 ITS rRNA基因扩增和系统发育分析 采用引物ITS1f (5′-CTTGGTCATTTAGAGG AAGTAA-3′)和ITS4 (5′-TCCTCCGCTTATTGA TATGC-3′)进行PCR扩增[9]。扩增体系：2×PCR Master Mix 12.5 μL，3 mmol/L MgCl2 2.0 μL，0.2 μmol/L primer 0.5 μL，DNA模板2 μL，加ddH2O补足至25 μL。热循环条件：94 ℃ 3 min；95 ℃ 30 s，55 ℃ 30 s，72 ℃ 30 s；40个循环，72 ℃ 6 min。PCR产物由上海生工生物科技有限公司采用ABI377 DNA自动测序仪直接测序。将所得的ITS基因序列采用BioEdit 7.0.5软件(Http://www.mbio.ncsu.edu/bioedit.ht)进行手动校正，统一序列方向和长度，输出FASTA格式的文件，在GenBank数据库中进行同源序列分析(BLAST)，构建系统发育树。用CLUSTAL X 1.83软件进行序列联配，采用neighbor-joining method计算进化距离，使用MEGA 6.0软件中p-distances和Kimura-2parameter双参数法构建系统发育树，并用Bootstrap检验进化树分支聚类的置信度，重复1000次。
1.8 最适温度测定 选取25株低温酵母作为代表菌株，经活化后以2%的接种量接到5 mL YEPG液体培养基中，设4、10、15、20、25、30、37 ℃ 7个温度梯度，培养箱中培养96 h，用紫外分光光度计于420 nm处测其OD值，根据溶液浓度判断其生长速率。
1.9 耐盐度的测定 制备初始NaCl浓度(W/V)分别为1.5%、3.0%、4.0%、5.0%、6.5%、8.0%的YEPG液体培养基。并以2.0%的接种量将已活化的酵母菌接到装有5 mL YEPG液体培养基的玻璃试管中，15 ℃培养96 h后，用紫外分光光度计于420 nm处测其OD值[21]。
1.10 产脂肪酶菌株的筛选及菌落特征 通过含有Yeast extract 10.0 g，Peptone 10.0 g，NaCl 5.0 g，CaCl2·H2O 0.1 g，Tween-80 10.0 g，琼脂16.0 g，pH 6.8的Tween-80琼脂培养基[22]检测脂肪酶活性，菌落周围出现明显的沉淀圈，则表明具有降解脂肪的能力，15 ℃培养96 h。
1.11 产蛋白酶菌株的筛选及菌落特征 通过以casein为底物的YEPG培养基[23] (含Skim milk 20 g/L，pH 6.5)进行蛋白酶活性检测，以菌落周围是否产生透明圈来表示蛋白酶活性，15 ℃培养96 h。
1.12 产淀粉酶菌株的筛选及菌落特征 在含有Soluble starch 20 g/L的YEPG培养基[24]中，向培养皿中加入稀碘液，以菌落周围出现明显的水解圈的大小，判断其水解淀粉的能力，15 ℃培养96 h。
2 结果和分析 2.1 乌鲁木齐天山一号冰川表面冰尘及底部沉积物的理化性质的比较 结果表明，天山一号冰川表面冰尘颜色为灰褐色颗粒物，且分布较为均匀，颗粒直径约为0.34-4.00 mm，pH约为7.0，冰川底部沉积层土壤pH约为7.8 (采样点见图 1)。冰尘样品(编号CS)中速效磷、速效钾、硝态氮、有机质以及全氮含量均显著高于冰川底部沉积层土壤样品(编号为DS)，表面冰尘的硝态氮及全氮含量是底部沉积层土壤的8-10倍，速效磷、速效钾及有机质含量达到3-5倍。只有全磷、全钾含量与冰川底部环境相近(表 1)。
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| 图 1 冰川表面(A)、冰川末端沉积物(B)及冰尘表面(C)取样点 Figure 1 Photographs showing the sampling sites on the glacier surface (A), subglacial sediments (B) and supraglacial cryoconite (C). |
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表 1. 冰川表面冰尘和底部沉积物的理化特性比较 Table 1. Physicochemical properties of supraglacial cryoconite and subglacial sediments
| Sample | SM*/% | pH(water：soil=2.5:1) | AP/(mg/kg) | AK/(mg/kg) | NO3--N/(mg/kg) | OM*/% | TN*/% | TP*/% | TK*/% | |
| Supraglacial cryoconite | Mean | 45.56 | 7.02 | 11.94 | 195.60 | 88.11 | 8.40 | 0.38 | 0.110 | 2.77 |
| SD | 12.45 | 0.45 | 3.46 | 56.68 | 32.48 | 4.34 | 0.07 | 0.035 | 0.54 | |
| Subglacial sediments | Mean | 32.45 | 7.75 | 2.57 | 144.50 | 8.90 | 1.37 | 0.03 | 0.072 | 3.55 |
| SD | 10.80 | 0.23 | 0.32 | 32.65 | 3.54 | 0.36 | 0.012 | 0.014 | 0.08 | |
| *: Physical and chemical characteristics under dry weight conditions. | ||||||||||
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2.2 冰尘及底部沉积物低温酵母的分离纯化 天山1号冰川冰尘和底部沉积层中分离纯化出152株可培养酵母菌，纯化后的菌株依据菌落颜色、细胞形态分类，菌落颜色丰富(图 2)，有白色、乳黄色、橘色、橘红色，少数为浅粉色、红色。酵母菌菌落直径约1.5-3.0 mm，小部分菌落直径小于1 mm，酵母菌的细胞形态(图 3)通常有球形、卵圆形、腊肠形、椭圆形，柠檬形或藕节形等，一般为2-4 μm。
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| 图 2 部分酵母菌菌落照片 Figure 2 Morphological characteristics of colony of some yeast strains. A: YD-15, Sporobolomyces ruberrimus; B: C8, Cystobasidium larynges; C: C9, Cystofilobasidium macerans; D: RS-5, Papiliotrema flavescens; E: C11, Dioszegia hungarica; F: YS-6, Leucosporidium muscorum; G: C53, Filobasidium stepposus; H: C80, Sporobolomyces metaroseus. |
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| 图 3 部分酵母菌细胞的显微结构 Figure 3 Optical microscope observation of vegetative cells of some yeast strains. A: DS-14, Naganishia albidus; B: C35 Vishniacozyma tephrensis; C: C2, Vishniacozyma victoriae; D: RD-6, Vishniacozyma carnescens. |
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2.3 基于ITS rRNA基因序列扩增及系统发育分析 扩增冰川表面冰尘和底部沉积物中已分离纯化的低温酵母菌菌株的ITS序列并测序，通过使用NCBI (National Center of Biotechnology Information)中的BLAST比对工具在GenBank数据库中进行同源性比对，将152株低温酵母菌进行归类划分(表 2)，图 4为CS(A)及DS(B)酵母菌属水平上的群落结构组成，同时选取25株代表酵母菌菌株的序列构建系统发育树(图 5)。白逢彦等[25]使用聚类优化分析法(Phylogenetic rank boundary optimisation，PRBO)和溯祖理论分析法(General mixed Yule coalescent，GMYC)以及贝叶斯法(Bayesian method)等分析方法，对银耳纲(Tremellomycetes)酵母菌进行重新归类(表 2)。由系统发育树可知，25株冰川低温酵母代表菌株分别隶属于担子菌门(Basidiomycota)和子囊菌门(Ascomycota) 2个门水平的系统发育类群。
表 2. 天山1号冰川不同生境中酵母菌种类的分布 Table 2. Occurrence and distribution of yeast species in different habitats of Glaciers No. 1 in the Tianshan Mountains
| Species | Basionym or important synonym | Class | Strain No. (accession number) | Similarity/% | Habitats (No. of isolates) | Total No. of isolates | |
| CS | DS | ||||||
| Basidiomycota | |||||||
| Agaricomycotina | |||||||
| Naganishia adeliensis | Cryptococcus adeliensis | Filobasidiales | C56(MF062219) | 99 | 8 | 8 | |
| Naganishia albidus | Cryptococcus albidus | Filobasidiales | DS-14(MF062213) | 100 | 1 | 1 | |
| Naganishia liquefaciens | Cryptococcus liquefaciens | Filobasidiales | C73(MF062229) | 100 | 6 | 3 | 9 |
| Filobasidium magnus | Cryptococcus magnus | Filobasidiales | YD-1(MF062227) | 99 | 1 | 2 | 3 |
| Filobasidium stepposus | Cryptococcus stepposus | Filobasidiales | C53(MF062197) | 99 | 2 | 2 | |
| Filobasidium elegan | Filobasidiales | YD-3(MF062225) | 100 | 2 | 2 | ||
| Filobasidium floriforme | Filobasidiales | C67(MF062187) | 99 | 1 | 1 | ||
| Papiliotrema flavescens | Cryptococcus flavescens | Tremellales | RS-5(MF062194) | 99 | 1 | 1 | |
| Vishniacozyma carnescens carnescens carnescens | Cryptococcus carnescens | Tremellales | RD-6(MF062205) | 99 | 4 | 4 | |
| Vishniacozyma tephrensis | Cryptococcus tephrensis | Tremellales | C35(MF062236) | 99 | 7 | 7 | |
| Vishniacozyma victoriae | Cryptococcus victoriae | Tremellales | C2(MF062235) | 100 | 26 | 12 | 38 |
| Dioszegia hungarica | Cryptococcus hungaricus | Tremellales | C11(MF062232) | 98 | 2 | 2 | |
| Cystofilobasidium magnus | Cryptococcus macerans | Cystofilobasidiales | C9(MF062233) | 99 | 1 | 1 | |
| Mrakia gelida | Cystofilobasidiales | YD-31 (MF062220) | 100 | 5 | 5 | ||
| Mrakia aquatica | Mrakiella aquatica | Cystofilobasidiales | C79(MF062228) | 99 | 3 | 3 | |
| Mrakia cryoconiti | Mrakiella cryoconiti | Cystofilobasidiales | RD-8(MF062226) | 99 | 1 | 1 | |
| Pucciniomycotina | |||||||
| Leucosporidium muscorum | Leucosporidiella muscorum | Microbotryales | YS-6(MF062190) | 99 | 1 | 1 | |
| Sporobolomyces metaroseus | Sporidiobolus sp. | Moniliales | C70(MF062230) | 99 | 5 | 1 | 6 |
| Sporobolomyces ruberrimus | Schizonellamela nogramma | Moniliales | YD-15(MF062202) | 100 | 2 | 2 | |
| Phenoliferia glacialis | Rhodotorula glacialis | Erythrobasidiale | C52(MF062201) | 99 | 2 | 1 | 3 |
| Cystobasidium laryngis | Rhodotorula laryngis | Erythrobasidiale | C8(MF062208) | 99 | 4 | 4 | 8 |
| Cystobasidium minuta | Rhodotorula minuta | Erythrobasidiale | C21(MF062212) | 99 | 1 | 3 | 4 |
| Ascomycota | |||||||
| Candida akabanensis | Gemmamycetes | DS-1(MF062215) | 99 | 19 | 19 | ||
| Candida rugosa | Gemmamycetes | DS-26(MF062223) | 99 | 18 | 18 | ||
| Yarrowia lipolytica | RD-15(MF062199) | 99 | 1 | 1 | |||
| Aureobasidium pullulans | C20(MF062189) | 100 | 1 | 1 | 2 | ||
| 70 | 82 | 152 | |||||
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| 图 4 冰川表面冰尘(A)及底部沉积层(B)酵母菌属水平上的群落结构组成 Figure 4 Relative abundance of the yeast genus form supraglacial cryoconite (A) and subglacial sediments (B). |
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| 图 5 基于ITS rRNA基因序列酵母菌系统发育树 Figure 5 Neighbour-joining tree showing the phylogenetic relationships among yeast strains ITS gene partial sequences and related sequences acquired from GenBank. Numbers at the nodes indicate the bootstrap values based on neighbour-joining analyses of 1000 resampled datasets. Bar 0.05 at the bottom is the sequence digergence. |
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统计显示(图 4)，Vishniacozyma victoriae占的比重最大，为优势菌株，其中占CS样品总菌株数的37.14%，占DS样品总菌数的14.63%。仅在DS样品中分离出Candida akabanensis和Candida rugosa，分别占DS样品总数的10.92%和10.34%。在2个样品(CS/DS)中均分离出来Naganishia liquefaciens (8.57%/3.66%)、Filobasidium magnus (1.43%/2.44%)、Sporobolomyces metaroseus (7.14%/1.22%)、Phenoliferia glacialis (2.86%/1.22%)、Cystobasidium larynges (5.71%/4.88%)、Cystobasidium minuta (1.43%/3.66%)和Aureobasidium pullulans (1.43%/1.22%)，仅在CS样品中分离出Naganishia adeliensis (11.43%)、Filobasidium stepposus (2.86%)、Filobasidium floriforme (1.43%)、Vishniacozyma tephrensis (10.00%)、Dioszegia hungarica (2.86%)、Cystofilobasidium macerans (1.43%)、Mrakia aquatic (4.23%)和Sporobolomyces metaroseus (7.14%)。在DS样品中仅分离得到Naganishia albidus (1.25%)、Filobasidium elegan (2.44%)、Papiliotrema flavescens (1.22%)、Vishniacozyma carnescens (4.88%)、Mrakia gelida (6.09%)、Mrakia cryoconiti (1.22%)、Leucosporidium muscorum (1.22%)、Sporobolomyces ruberrimus (2.44%)和Yarrowia lipolytica (1.22%)。
对低温酵母代表菌株进行ITS基因序列分析，由图 5可知，伞菌亚门有18株菌，其中C9与C. macerans S3同源性高达100%，亲缘关系最近；C7、RD-8和YS-31分属于同一个进化支，与Mrakia同源性最高；C73、DS-14和C56分属于同一个进化支，与Naganishia同源性最高，相似度为99%；C67、YD-1、YD-3、C53聚为一类，与Filobasidium亲缘关系最近；菌株RS-5与P. flavescens AUMC7794相似度为100%；C11单独成为一个分支，与D. hungarica CBS 5124同源性最高，相似度为100%；C11、YD-15和C20分类成若干个小分支，分别与Dioszegia、Aureobasidium和Yarrowia亲缘关系最近。底部沉积层中分离得到的菌株YS-6与L. muscorum YFB256同源性最高，相似度为99%，与冰尘中分离出的C8、C21、C52均被归类在柄锈菌亚门。RD-15、DS-1和DS-26均从底部沉积层中分离出来，并聚为一类，隶属于子囊菌门。
2.4 冰川酵母菌的最适生长温度与耐盐度 实验结果表明(表 3)，25株低温酵母中2株菌的最适生长温度为10 ℃，17株最适生长温度为15 ℃，6菌株最适生长温度为20 ℃，56%的菌株在25 ℃停止生长。耐盐实验中，只有C52的耐盐度最低，84%的菌株耐盐度为6.5%，C67、YS-6、DS-26的耐盐度最高，在8%的盐浓度下也可缓慢生长。
表 3. 天山1号冰川部分低温酵母菌形态特征、生长特性及产酶活性 Table 3. Growth characteristics and morphological characteristics and extracellular enzymatic activity of partial cold-adapted yeasts difference habitats of Glacier No.1 in the Tianshan Mountains
| Strain | Morphological properties | Growth characteristics | Enzymatic activity | ||||||
| Colony color | Colony size/mm | Range temp/ ℃ | Range NaCl/% | Protease activity | Lipase activity | Amylase activity | |||
| C56 | White | 1-2 | 4-20a-30 | 0-6.5 | - | - | - | ||
| DS-14 | Pink | 2-3 | 4-15-30 | 0-6.5 | - | - | + | ||
| C73 | White | 2-4 | 4-15-25 | 0-6.5 | +++ | - | - | ||
| YD-1 | White | 2-4 | 4-15-25 | 0-6.5 | ++ | ++ | ++ | ||
| C53 | white | 1-2 | 4-15-25 | 0-4.0 | - | ++ | - | ||
| YD-3 | Yellow | 3-4 | 4-20-25 | 0-6.5 | - | - | - | ||
| C67 | yellow | 2-3 | 4-15-30 | 0-8.0 | - | - | - | ||
| RS-5 | Yellow | 2-3 | 4-15-25 | 0-6.5 | - | - | +++ | ||
| RD-6 | White | 1-2 | 4-20-25 | 0-6.5 | - | ++ | + | ||
| C35 | yellow | 2-3 | 4-15-25 | 0-6.5 | - | ++ | ++ | ||
| C2 | yellow | 2-3 | 4-15-30 | 0-6.5 | - | ++ | - | ||
| C11 | Orange | 3-4 | 4-15-30 | 0-6.5 | - | + | - | ||
| C9 | Pink | 2-4 | 4-20-30 | 0-6.5 | - | +++ | - | ||
| YD-31 | White | 1-2 | 4-15-20 | 0-6.5 | - | - | - | ||
| C79 | White | 2-3 | 4-15-25 | 0-6.5 | - | - | ++ | ||
| RD-8 | Yellow | 2-3 | 4-10-25 | 0-6.5 | - | + | + | ||
| YS-6 | Yellow | 3-4 | 4-20-30 | 0-8.0 | - | - | ++ | ||
| YD-15 | Yellow | 2-3 | 4-15-30 | 0-6.5 | - | - | + | ||
| C52 | White | 3-5 | 4-15-25 | 0-6.5 | +++ | +++ | - | ||
| C8 | Pink | 3-4 | 4-15-25 | 0-6.5 | - | - | - | ||
| C21 | Pink | 3-4 | 4-10-25 | 0-6.5 | - | - | - | ||
| DS-1 | Yellow | 2-3 | 4-15-25 | 0-6.5 | - | - | ++ | ||
| DS-26 | Orange | 2-3 | 4-15-20 | 0-8.0 | +++ | - | - | ||
| RD-15 | White | 2-3 | 10-20-30 | 0-6.5 | ++ | + | +++ | ||
| C20 | White | 1-2 | 4-15-25 | 0-6.5 | - | +++ | - | ||
| +: diameter 5-9 mm; ++: diameter 10-15 mm; +++: diameter ≥1.6 cm. a: The optimal growth temperature. | |||||||||
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2.5 酵母菌产酶活性及产酶菌株的形态特征 25株低温酵母产酶结果显示(表 3)，11株产淀粉酶，11株产脂肪酶，5株产蛋白酶。产蛋白酶菌株中有3株产酶活性最高，分别为C73、C52、DS-26，测得它们菌落周围沉淀圈和透明圈直径较大。产脂肪酶的菌株中有3株产酶活性最高，分别为C9、C52、C20，C20产酶的透明圈最为明显。在产淀粉酶菌株中，RS-5、RD-15这2株产酶活性最高，菌落周围沉淀圈和透明圈直径都超过了16 mm。其中YD-1和RD-15均产脂肪酶、蛋白酶和淀粉酶。RD-6、C35、RD-8和C52只产其中的2种酶。其余的菌株均只产其中的一种酶或不产酶。产酶菌落形态见图 6。
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| 图 6 菌株产胞外酶图片 Figure 6 Photographs of yeast strains producing extracellular enzymes. Protease activity: A: C73, Naganishia liquefaciens; B: RD-15, Yarrowia lipolytica; C: C52, Phenoliferia glacialis. Lipase activity: D: C9, Cystofilobasidium magnus; E: C2, Vishniacozyma victoriae; F: C20, Aureobasidium pullulans. Amylase activity: H: RS-5, Papiliotrema flavescens; I: RD-6, Vishniacozyma carnescens; G: RD-15, Yarrowia lipolytica. |
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3 讨论 冰尘是由有机物质、无机矿物颗粒及微生物结合形成的黑色或棕色球状聚合体。冰尘中存在大量的有机物质主要是自养型微生物的有机产物、冰川上的生物残体和风携带来的土壤中的有机物质[26]。冰尘吸收太阳辐射能量后，在冰川表面形成冰尘空洞，冰尘空洞中有机质含量丰富，栖息着大量的自养、异养微生物，如作为初级生产者的光合微生物(蓝绿藻、蓝细菌)等[27]。冰川表面的光自养微生物只能提供少量的有机碳和氮养分，冰川表面相对充足的养分大部分来自外源空气中颗粒的沉降积累作用[28]。冰川表面积雪、冰层的溶解，使部分融水通过冰川表面形成的锅穴和裂隙到达冰川底部，碳、氮养分也随融水到达冰川底部。本研究结果显示，冰川表面冰尘中硝态氮及全氮含量是底部沉积层土壤的8-10倍，速效磷、速效钾及有机质含量达到3-5倍。广泛存在于自然界中的真菌作为生态系统中的分解者，分解有机物质获得能量和营养物质，介导氮、磷循环，为异养、寄生微生物的生长繁殖提供养分[29]。
本实验通过使用4种不同的分离培养基将表面冰尘和底部沉积层2个样品中的低温酵母进行分离纯化，获得了152株可培养低温酵母，包括Vishniacozyma、Mrakia、Rhodotorula、Aureobasidium、Candida、Dioszegia、Cystofilobasidium和Sporobolomyces等。Zalar等[30]在阿根廷巴塔哥尼亚和挪威斯瓦尔巴德岛的冰川分离出的最优势担子酵母菌为V. victoriae。这与我们实验分离到的优势菌株结果一致。另外，V. victoriae在其他极端生境中也广泛生存，如南极洲南部维多利亚地区的土壤[31]、北极冰川表面冰[32]以及葡萄牙的深海[33]等。这说明V. victoriae生态分布非常广泛多样，具有极强的生存能力和定植能力。因此，对天山1号冰川V. victoriae的遗传分化以及生态位适应性进一步深入研究是非常关键的，另撰文报道。
Buzzini等[1]对欧洲、亚洲、南北两极的冰川表面及底部沉积物的酵母菌已进行了广泛的研究报道，最常见的酵母菌包括Cryptococcus、Mrakia、Rhodotorula、Candida和Dioszegia等，本实验也同样检测到这些菌种。Singh等[34]报道了从北极斯瓦尔巴德岛上Midre Lovénbreen冰川分离到Cryptococcus、Mrakia、Rhodotorula、Phialophora和Articulospora等。有研究表明，Articulospora和Varicosporium在冰尘孔碳循环过程中起积极作用[35]。奇怪的是Phialophora和Articulospora却未能在天山1号冰川中分离到。我们认为这可能是有于地理隔离等原因造成，不同地区环境的差异、多变的季节、水分的活度和理化特性的改变都会影响酵母菌的分布。
嗜冷菌分为专性嗜冷菌和兼性嗜冷菌，专性嗜冷菌的最适生长温度为15 ℃，最高生长温度为20 ℃，在0 ℃或更低的温度下也会缓慢生长，兼性嗜冷菌的最适生长温度为20-30 ℃之间[36]。对25株低温酵母进行了生态、生理实验，结果表明，2个样品中分离出的大部分菌株都为专性嗜冷菌，只有从冰尘中分离出的2个菌株C56、C9和从底部沉积层中分离出的4个菌株RD-6、YD-3、YS-6、RD-15为兼性嗜冷菌。研究表明，冰尘孔中丝状真菌和酵母菌等异养有机体，通过分泌胞外水解冷适应酶分解有机物，促进养分循环，使其能够在寡营养环境中生存[34]。Purnima等[5]对Austre Br?ggerbreen冰川和Vestre Br?ggerbreen冰川分离出的Articulospora、Thelebolus和Rhodotorula这3个属进行了产酶分析，表明其生态作用是通过自身产酶机制对有机高分子分解进行营养循环，其中Articulospora和Rhodotorula产脂肪酶和淀粉酶的活性很高。本实验结果表明，Yarrowia和Filobasidium这2个属的菌株均产脂肪酶、蛋白酶和淀粉酶，Naganishia、Rhodotorula和Candida这3个属产蛋白酶的活性很高，Articulospora产脂肪酶的活性最好，Papiliotrema和Yarrowia两个属产淀粉酶的活性最好。天山1号冰川分离出的低温酵母菌颜色丰富多样，形态大小不一。酵母菌通过改变极性和非极性类胡萝卜素来调节细胞膜的流动性[37]，从而更好地适应低温环境。Zawierucha等[38]发现，低温微生物色素沉着可以保护自身免受高强度紫外线辐射带来的损伤，深色色素同时也会吸收大量的热量，导致冰川融化速度加快。低温微生物是冰川生态系统中重要的组成部分，因此深入研究冰川低温微生物，分析低温微生物的遗传多样性、生理生化特性及其生态位，有重要的科学意义。
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