海洋生物固氮研究进展

李志红, 李劲尤, 刘甲星

海洋生物固氮研究进展

李志红1, 李劲尤2, 刘甲星3, 4, *

1. 珠海万山海洋开发试验区海洋渔业科技发展促进中心, 珠海 519005 2. 香港城市大学, 数据科学学院, 香港 3. 中国科学院南海海洋研究所, 海洋生物资源可持续利用重点实验室, 广州 510301 4. 南方海洋科学与工程广东省实验室(广州), 广州 511458

海洋生物固氮因可以支持初级生产所需的氮而在全球碳氮循环中具有重要作用。从二十世纪九十年代分子生物学和15N2同位素示踪法应用于固氮研究领域以来, 逐渐发现了单细胞固氮蓝藻和异养固氮细菌的重要性, 是近年来海洋固氮研究领域的最大进展之一, 表明以前基于束毛藻为主要固氮生物估算的固氮量可能低估了生物固氮在全球海洋生物地球化学循环中的地位。另一方面, 传统的海洋生物固氮研究仅局限于热带亚热带的寡营养盐区域, 对高营养盐区域如上升流、河口等高营养盐区域较少关注, 因此有必要对这些区域的生物固氮进行重新评估和再认识。综述了国际固氮研究的最近进展, 主要包括固氮生物多样性及分布特征、生物固氮的限制性因素、研究方法以及存在的问题。同时综述了南海生物固氮方面的最新进展和问题。

海洋生物固氮; 单细胞固氮蓝藻; 异养固氮细菌; 分子生物学技术; 高营养区域; 南海

0 前言

海洋固氮生物可以利用氮气转化为生物可利用氮, 这一过程叫作生物固氮, 每年固定的新氮(大约100—150 Tg)占全球氮输出总量一半以上[1]。在贫营养环境中, 生物固氮固定的氮是上层海洋新氮的重要来源, 可以有效缓解上层海洋的氮限制[2]。同时, 对于二氧化碳的净吸收也起着重要作用[3]。因此, 海洋生物固氮在海洋碳氮循环中发挥着重要作用, 是国际研究的热点。

自1961年在马尾藻海发现束毛藻可以固氮以来[4], 由于研究方法的限制, 人们一直认为束毛藻是最重要的海洋固氮生物。直到20世纪90年代分子生物学技术和15N2同位素标记法应用于海洋生物固氮研究, 在对固氮生物种类和分布的认识上取得了一系列突破性的进展, 每年发表的研究论文逐渐增加(图1a, 表1)。如Zehr等(2001)发现了单细胞固氮蓝藻在海洋生物固氮中也起着同样重要的作用, 随后也发现异养固氮细菌在某些海域固氮生物群落中占据优势[5–6], 拓展了人们对于固氮生物的认识[7]。尽管国际上海洋生物固氮相关的研究已经相当多, 但是依然有很多问题尚待探究。相对而言, 中国边缘海的海洋生物固氮研究薄弱, 直到2000年以后才逐渐有一些研究报道(图1b), 研究的深度和广度明显落后于其他国家。本文在综述国际上生物固氮领域的最新进展的同时, 以南海为例, 介绍了我国固氮研究的现状和存在的问题, 期望为中国边缘海固氮研究做参考。

1 海洋固氮生物多样性

海洋固氮生物主要包括固氮蓝藻和固氮异养细菌, 目前认为固氮蓝藻是海洋中主要的固氮生物[1, 24], 包括丝状固氮蓝藻、共生固氮蓝藻和单细胞固氮蓝藻[25]。其中, 束毛藻(Trichodesmium spp.)是最常见且最重要的一类丝状固氮蓝藻[4, 26], 主要生活在水温高于20℃的寡营养海域上层[27], 在24℃—30℃度之间最适合固氮, 易在海洋表层形成赤潮[26]。束毛藻固氮具有一定的节律性, 固氮发生在白天。目前发现的共生固氮蓝藻主要有: 与根管藻(Rhizosolenia spp.)和半管藻(Hemiaulus spp.)共生的Richelia(分别为Het-1和Het-2), 以及与角毛藻(Chaetocerous)共生的Calothrix (Het-3)28–29], 这些固氮蓝藻喜欢生活在营养丰富的近岸区域[7, 29-36]。单细胞固氮蓝藻个体一般小于10μm, 在寡营养和富营养区域都有广泛分布, 且在深水层仍有较高丰度, 其固氮量可达海洋固氮总量的30%—70%, 在某些区域超过束毛藻[37], 主要分为三个类群(UCYN-A, UCYN-B和UCYN-C)[15, 38–40]。UCYN-A尚未得到室内培养, 直径小于1μm, 固氮发生在白天, 生长温度范围为15–30°C, 不能独立进行光合作用[41], 与进行共生[42–43]; UCYN-B以可在室内培养的为代表, 细胞呈球形, 直径范围为3—10μm, 可以独立进行光合作用, 固氮一般在晚上进行, 最适生长温度为22—36°C[15, 38, 44–45]; UCYN-C以可培养的为代表, 可以进行光合作用, 固氮也在晚上发生[46]。此外, 也发现一些异养细菌含有固氮基因(), 如α、β和γ变形菌等[6, 7, 47–50], 尽管在南太平洋、印度洋和南海北部区域报道了异养细菌是主要的固氮生物[5–6, 51–53],但是其固氮活性重要性尚无法判断[7]。

图1 Web of science检索到的海洋生物固氮年发表文章数(a)与国家发表论文数(b)

Figure 1 Number of published articles on marine nitrogen fixation every year (a) and national publications (b) retrieved by web of science

表1 海洋生物固氮研究历史中的重要事件

2 海洋生物固氮的影响因素

2.1 温度、盐度、光和氧气

温度可以影响固氮生物的生长和固氮, 因此影响着固氮生物的分布[54–55]。野外调查的结果表明, 束毛藻可以生活在20—30℃之间的海域, 最适温度为25—30℃[26–27, 56–57]; 室内培养实验也表明, 20—34℃为束毛藻生长和固氮的温度范围, 最适范围为24—30℃, 在27℃时最活跃[58]。单细胞固氮类群似乎更能耐受更低的温度, 如可以生活在18—30℃[59], 在温度较低区域也很常见[39–40]。另外, 盐度也可以影响固氮生物生长和固氮, 束毛藻有较广泛盐度耐受性(22—43), 33—37是最适固氮盐度区间[60]; 与共生固氮蓝藻可以在较低的盐度生存, 所以经常在河口等近岸[29, 61]。

光可以影响固氮酶的活性, 同时也影响细胞的光合作用, 因此也能影响固氮生物的生长和固氮[62]。束毛藻一般在表层丰度较高, 因为其对光的需求较高, 光强增加可以促进固氮速率增加[63], 西太平洋的现场培养实验表明束毛藻的光饱和强度在400 μmolquanta·m-2·s-1左右[64]; 室内实验也表明, 束毛藻在670 μmol quanta·m-2·s-1光照强度下还可以快速生长(生长速率可以达到0.49 d-1)[65]。光强对单细胞固氮蓝藻同样也具有一定的影响, 如室内实验表明Crocosphaera在180 μmol quanta·m-2·s-1光强下生长速率和固氮速率随着光照强度增加而增加, 在300 μmol quanta·m-2·s-1光照条件下生长速率和固氮速率没有受到明显抑制[66]; 当温度在14-30°C范围内时,在60 μmol quanta·m-2·s-1可以达到光饱和状态[59]。另外, 光照周期也会对固氮蓝藻造成影响, 12h光照/12h黑暗的光照周期条件下Cyanothece固氮速率最大, 延长或者缩短光照时间都明显抑制固氮速率[67]。除了光强和光周期, 不同光谱也会影响固氮生物的生长, 室内培养实验表明紫外线可以抑制束毛藻生长和固氮, 但束毛藻在受到紫外线激发时产生类菌胞素氨基酸MAAs以减少紫外线的抑制作用[68]。

此外, 氧气也能抑制固氮酶活性, 因此固氮生物采取多种机制来避免光合作用产生的氧气对生物固氮的抑制[1, 69–70]。具有异形胞的丝状固氮蓝藻固氮作用发生在异形胞内部, 异形胞是分化的细胞, 不能进行光合作用, 但是可以进行固氮作用, 这一特定分化的细胞使整个丝状固氮蓝藻的光合作用和固氮作用形成区域分割, 有效的避免了氧气对固氮酶的抑制[26]。没有异形胞的丝状蓝藻束毛藻, 也可以在白天进行固氮作用, 可能与其独特的细胞结构或者固氮酶特征有关[26]。对于单细胞固氮蓝藻, UCYN-A细胞营共生生活, 由于细胞缺乏光系统而不能进行光合作用, 因此在白天进行固氮[41]; 而对于UCYN-B和UCYN-C而言, 固氮光合作用在晚上发生, 以有效避免氧气对固氮酶的抑制[15, 38, 44–46]。

2.2 营养元素

铁参与固氮酶复合体的形成, 因此, 固氮生物对铁的需求量要高于其他浮游植物[71]。在寡营养的大洋区域, 铁主要来源于大气沉降和水体的垂直或水平输送, 大气干湿沉降的铁对上层水体的固氮生物生长和固氮起着至关重要的作用[72]。已经在一些区域得到证实, 如南大西洋固氮速率与溶解性铁成正相关而与磷负相关[73]; 在边缘海也有铁限制固氮生物的报道, 如在南海海盆区域, 尽管大气沉降带来大量铁, 但依然存在缺乏配体而导致生物可利用铁缺乏[74]; 基于原位分子生物学的结果也表明大西洋和太平洋一些区域铁是束毛藻生长和固氮的限制性元素[75]。原位培养实验也表明, 铁是西北大西洋固氮生物,和固氮的限制性因素[76]。室内培养实验也发现铁缺乏可抑制束毛藻固氮[77–79], 缺铁也可以对生长和固氮产生不利影响[80–81]。

磷在参与细胞构建以及核酸、蛋白质等大分子物质合成、能量传递等方面起着重要作用, 同时也对固氮生物的生长和固氮等生理过程具有重要影响。很多原位观测结果证实了这一结论, 如基于无机磷浓度的结果表明西北大西洋上层固氮生物生长可能受到磷限制[82]; 基于碱性磷酸酶和固氮速率的直接证据表明北大西洋海盆区域束毛藻生长受到磷限制[83]; 基于碱性磷酸酶、铁结合蛋白基因和固氮速率的观测结果表明西南大西洋生物固氮的限制性因子可能为磷[84]; 基于固氮速率和束毛藻丰度的结果表明南海琼东上升流影响区域磷可能是生物固氮的限制性因子[85]。同时, 原位培养实验的结果也证明了许多区域存在磷限制, 如波罗的海添加磷促进水体总固氮速率[86], 西南大西洋的实验表明添加磷也有效促进了束毛藻的生长和固氮[84], 东北大西洋区域的现场加富实验表明该区域磷是限制固氮生物,和固氮的主要因素[76]。为了适应磷匮乏的环境, 固氮生物进化了许多生存策略, 如束毛藻可以通过调节浮力在上层水柱进行垂直迁移获得磷[87], 同时可以在环境磷浓度低时高表达高亲和磷蛋白等相关基因以高效获取磷, 此外, 束毛藻具有分解有机磷的相关基因如和家族基因, 通过高表达碱性磷酸酶等水解有机磷来满足其生存[88]; UCYN-B也具有高亲临蛋白基因和分解有机磷相应的基因来应对磷缺乏[89–90]; 尽管UCYN-A基因组缺乏分解有机磷相关的基因和代谢途径[43], 但是其能与单细胞藻类进行共生[42–43], 这种关系可以使UCYN-A在低磷环境中生存[76]。

此外, 某些区域也存在磷和铁协同限制固氮生物生长的情况, 如北大西洋的现场加富实验表明, 固氮作用受到磷和铁的协同限制, 撒哈拉沙尘所携带的大量磷和铁共同刺激了该海域的固氮作用[91], 北大西洋赤道附近区域的现场加富实验也表明该区域磷和铁可能是协同限制固氮生物,和固氮[76]。

尽管钼也是固氮酶复合体的一个重要组成元素[92], 但在原位环境中报道钼限制固氮生物生长的研究较少, 可能是因为自然海水中具有较高的钼。此外, 溶解无机氮也会影响固氮生物固氮[1], 如束毛藻和也可以利用无机氮源, 但是固氮会被抑制[93–94]。另外, 也有研究表明环境中氮磷的比例也会对固氮造成一定影响[95–96]。

2.3 物理过程

2.3.1 中尺度涡

中尺度涡是一种重要的海洋动力学现象, 常见的中尺度涡旋分为三类, 气旋冷涡(cyclonic eddy), 反气旋暖涡(anticyclonic eddy)和模式水涡(Mode- water eddy), 其中冷涡一般导致中心水团向上涌升, 暖涡导致中心水团向下沉降, 模式水涡在一般导致中心水团上层涌升而下层沉降[97]。大量研究表明中尺度涡可以通过垂直输运水团改变水体的理化特征和营养盐等来影响浮游植物群落组成, 因而在全球碳循环中发挥着重要作用[98–99]。一方面, 中尺度涡可以通过物理混合作用影响固氮生物, 如水团混合导致束毛藻固氮酶活降低[100], 水体层化系数与束毛藻丰度负相关[101]。另一方面, 中尺度涡可以通过影响温度、盐度、营养盐等环境因子来调控固氮生物的分布及其固氮(表2)。已有不少研究表明反气旋涡可以促进固氮, 如北大西洋反气旋涡内束毛藻丰度较高[102–103], 北太平洋反气旋涡内固氮蓝藻(spp.,和)的丰度及总固氮速率都高于临近海域[104–105], 澳大利亚西海岸反气旋涡内固氮速率是气旋涡内的2.5倍[106], 地中海区域反气旋涡内固氮速率是临近海域的10倍[107], 南太平洋的研究也表明反气旋涡影响区域最大固氮速率出现在真光层以下, 且反气旋涡刺激的固氮作用在向近岸区域输送氮方面起着重要作用[108], 南海东北部暖涡区域固氮速率显著高于临近参考区域和冷涡区域, 同时UCYN-A和UCYN-B丰度较高[109]。相对而言, 冷涡区域的固氮报道的较少, 有限的结果表明冷涡也可以影响固氮, 如南太平洋的研究也表明, 气旋涡影响区域固氮仅发生在表层[108], 南海东北部冷涡区域固氮速率和固氮蓝藻丰度(UCYN-B 和Trichodesmium)低于临近区域[109], 南海海盆区气旋涡区域发现高丰度的固氮异养细菌[110]。这些研究表明中尺度涡可能在影响生物固氮方面起着重要作用, 同时可以明显调控固氮对初级生产力所需氮的贡献, 如南海东北部反气旋涡可以显著刺激生物固氮速率, 反气旋涡内固氮对初级生产力所需氮的贡献率可以达到9%左右[109]。

2.3.2 上升流等高营养盐区域生物固氮

沿岸上升流作为一种重要的中尺度物理现象, 其可以通过改变营养盐和浮游植物群落结构而对海洋生态系统具有重要影响[113-114]。一般认为, 沿岸上升流区域不存在或具有较弱的生物固氮作用。首先, 沿岸上升流区域有较高的氨氮和硝氮, 固氮生物不会耗费大量能量固氮(固氮过程需要消耗大量能量)[115]; 其次, 固氮生物在与其他浮游植物生长竞争中也常处于劣势地位[116]; 另外, 上升流区较低的温度似乎也不适合喜高温环境的固氮生物生长[1]。因此, 沿岸上升流区域的生物固氮研究一直没有得到学者们的重视。然而, 随后在如亚马逊河口和湄公河口等高营养区域也发现较强的固氮作用[31, 61]。一般认为固氮生物喜欢在高温高盐寡营养盐的低纬度地区, 然而在上升流、河口及冲淡羽等富营养盐海区也发现生物固氮相当活跃[18, 29, 31, 34–35, 40, 106, 116–121]。

尽管已有少量研究报道上升流对固氮生物及其固氮的影响, 然而, 对上升流区域的生物固氮分布模式及其形成机制的认识仍然十分有限。首先, 上升流区域是否存在生物固氮作用依然存在争议, 如在南太平洋的本格拉(Benguela)上升流区域检测到较高的固氮速率[25]; 但另外的研究认为, 该区域在上升流季节并不存在生物固氮现象, 原位培养实验也证实了这一结论[120]。其次, 上升流对固氮生物生长及其固氮影响的机制存在争议, 且固氮生物群结构落数据相对缺乏。生物固氮一般受到磷或铁的限制, 上升流带来的磷或铁可以刺激固氮生物生长及其固氮, 如在越南沿岸上升流和湄公河带来的大量磷和金属使该区域出现高固氮速率, 但没有直接检测固氮生物群落结构[18]; 大西洋赤道上升流带来的磷和铁可以促进该区域的生物固氮, 推测单细胞固氮蓝藻对该区域固氮具有明显贡献, 但也没有直接检测固氮生物类群[116]; 在南海的琼东上升流区域, 相对较高的固氮速率可能与上升流带来的磷和铁有关[85]。另一些研究认为上升流区域出现高固氮速率可能与有机物质和低氧有关。如在南太平洋上升流区域, 400米处依然检测到生物固氮作用, 且缺氧的次表层固氮速率是真光层的5倍之多, 在上层水体中没有发现固氮蓝藻, 却存在大量异养固氮细菌, 低氧可能是该区域高固氮的重要因素[122]; 在东南太平洋的智利上升流区域, 低氧和有机物质可能是上升流区域出现固氮的主要因素, 同时浮游植物群落结构及其碳氮比也会影响固氮速率, 且异养固氮细菌对该区固氮起着重要作用[123]。这些结果表明上升流对生物固氮影响的机制尚不清楚, 值得继续探究。

表2 中尺度涡对固氮生物群落结构和生物固氮速率影响的报道

3 海洋生物固氮的研究方法

早在1961年, Dugdale等就利用15N2示踪培养法发现束毛藻具有固氮活性[4]。通过添加15N标记的氮气, 利用元素同位素质谱仪检测培养后的颗粒有机氮, 就能示踪结合到颗粒有机氮的15N, 进而可以计算生物固氮速率。该方法灵敏度高, 是目前检测海洋固氮速率的常用方法[13]。该方法最早通过向培养体系中直接添加15N同位素标记的氮气来实现, 被称为气泡法[13], 后来有研究表明气泡法的结果可能低估了实际固氮速率[21, 124], 因此又发展出了海水添加法[125]。

Dilworth等在1966年发现固氮酶可以将乙炔还原为乙烯[9], 随后Stewart等在1967年将乙炔还原法扩大应用于湖泊等野外环境中[8], 此后该方法一直沿用至今。该方法的原理为, 在同时存在乙炔和氮气的情况下, 固氮酶优先把乙炔还原为乙烯[9], 一般认为被还原的乙烯与被固定的氮气存在固定的比例即3:1, 以此便可以估算出固氮速率[8]。但是该方法的也有一定的局限性[126], 因为不同环境下乙烯和氮气之间的比值不尽相同[127]。

尽管同位素法和乙炔还原法可以有效的检测环境中的总固氮速率, 但是随着越来越多的固氮生物被发现, 检测环境中每个类群的固氮活性已经成为迫切。自1989年Zehr等利用PCR技术首次扩增了束毛藻基因以来, 分子生物学方法逐渐应用到海洋生物固氮的研究中。固氮酶一般由含铁还原酶(N2reducatse)和含钼铁固氮酶(N2dinitrogenase)组成, 分别由和基因编码, 此外还有一些含钒离子的固氮酶[128], 其中基因序列高度保守, 功能信息比的更丰富, 且其表达受到时间、空间、光照和营养盐影响, 因此序列是鉴定固氮生物物种及分析固氮活性的重要依据[129]。大部分固氮生物的拷贝数目相对稳定, 可以基于的拷贝数来估算某一类群固氮生物的丰度和揭示固氮生物群落结构信息[40–41]。同时, 利用基因表达的水平可以推测某一类固氮生物类群的固氮活性。因此分子生物学方法是研究固氮生物群落结构及固氮活性的好方法[130], 尤其随着定量PCR技术、二代测序技术、基因组、转录组和蛋白质组技术的发展, 基于海量数据的大数据分析可以得到更加全面丰富的信息, 以便于从基因水平上揭示固氮生物对环境的适应性机制, 该方法是目前研究固氮生物的重要方法。

分子生物学方法尽管可以检测原位环境中固氮基因或者固氮基因表达量, 然而固氮基因表达量在某些情况下并不代表生物固氮的活跃程度, 因此仍然不能直接证明原位环境中的固氮量问题。荧光原位杂交-纳米二次离子质谱技术(FISH-NanoSIMS), 即把荧光原位杂交技术与二次离子质谱技术联用能够实现在原位上同时观察自然环境样品中微生物的形态结构、种类分布及功能代谢信息[131]。近年来已经普遍用于定量单细胞固氮速率和共生固氮固定氮转移问题的研究中, 展现了研究原位环境固氮的强大优势[33, 42]。

4 南海生物固氮的研究现状

南海大部分区域具有强层化、寡营养、温暖和强大气沉降的特征, 具有利于固氮生物生长的良好环境[25, 74]。氮是限制南海外海浮游植物生长的关键因子[132], 因此固氮对南海的碳氮生物地球化学循环具有重要作用。尽管南海生物固氮的研究起步较晚, 但近年来取得了一系列新成果。

在对固氮生物多样性的认识上, 束毛藻是南海常见的固氮类群, 普遍分布于珠江口影响区域[133]、三亚湾[134]、大亚湾[135]、琼东上升流区[85]、粤东上升流区[136]、湄公河河口和越南沿岸等近岸区域[18, 61, 137–138], 也在吕宋海峡及邻近南海海盆[109, 139–141]和南沙岛礁等离岸区域广泛存在[142], 同时在某些区域如大亚湾[135, 143]和粤东沿岸区域[85]附近经常发生束毛藻赤潮; 单细胞固氮蓝藻UCYN-A和UCYN-B在南海东北部区域普遍存在[96, 109, 133, 141, 144], 在吕宋海峡区域附近也发现了较高丰度且对总固氮量的贡献可达65%以上[145]; 在珠江口(Het-1)[133]、粤东上升流(Het-1)[96]和湄公河河口(Het-1和het-2)[18, 61, 138]等近海域存在与硅藻共生的固氮生物; 另外, 异养固氮细菌也广泛存在于大亚湾、南海北部和海盆区域[109, 133, 146–149]。

相对于固氮生物的研究而言, 直接检测固氮速率的研究相对较少[32]。已有发表的结果表明, 台湾海峡为11—40 μmol N·m-2·d-1[150], 秋季南海东北部近岸区域表层固氮速率范围为4—213 nmol N·m–3·h–1[143], 大亚湾四个季节固氮速率为0—4.51 nmol N·L-1·h-1[149], 夏季琼东上升流区域为7.5—163 μmol N·m-2·d-1[85], 夏季粤东上升流区域为0—7.51 nmol N·L-1·d-1[136], 南海东北部海盆区四个季节固氮速率为2.4—168.1 μmol N·m-2·d-1, 夏季出现最高固氮速率, 其次为春季和秋季, 冬季固氮速率最低[101, 109]。湄公河河口固氮速率为5.05—22.77 nmol N·L-1·h-1[61], 越南沿岸区域固氮速率为1.9—190.6 μmol N·m-2·d-1[18]。

在固氮生物分布的区域上, 除了传统上认为适合固氮生物生活的高温高盐寡营养盐的低纬度区域发现固氮生物以外, 也在河口、上升流和海湾等高营养也存在较高丰度的固氮生物, 如在湄公河河口束毛藻和硅藻共生固氮藻(Het-1和Het-2)出现高丰度和高固氮量[61, 137–138]; 在珠江口影响区域, 束毛藻在靠近外海站位相对较多[133], 同时珠江口影响的局部区域发生束毛藻赤潮和高固氮速率[143]。在越南沿岸上升流区域, 且在季风期间固氮速率是季风转换期间固氮速率的10倍左右[18]; 在琼东上升流区域也发现活跃的生物固氮作用, 上升流带来的铁可能刺激该区域固氮, 且局部出现束毛藻赤潮[85]; 在粤东上升流, 也发现上升流影响区域存在活跃固氮能力, 同时发现大量固氮蓝藻(束毛藻、UCYN-A和Het-1)[96]。同时, 在营养丰富的大亚湾等水域也经常会发生固氮生物束毛藻赤潮现象, 如1987年8月大亚湾海域的发生大面积束毛藻赤潮; 2004年6月大亚湾湾口发生束毛藻赤潮, 呈黄褐色“彩带”, 宽3海里、长8海里, 面积约为100平方公里[135]; 2007年深圳大鹏湾梅沙赤潮, 主要为汉氏束毛藻[151]; 2016年大亚湾湾外也检测到肉眼可见的束毛藻赤潮, 同时实测固氮速率异常高[143]; 大亚湾开展的研究也同时表明大亚湾区域高营养区域并没有限制固氮速率, 在某些区域反而出现较高固氮速率[149]。以上结果表明, 在高营养的近岸区域也会发生明显生物固氮现象, 与以往固氮生物尤其固氮蓝藻喜欢生活在贫乏营养状况下的结论不一致, 值得深入探究。

在对固氮生物生长限制性因子的认识上, 相关研究较少且存在争议。在南海海盆区尽管大气沉降可以输送充足的铁, 但是由于铁配体不足的致使生物可利用铁浓度较低(溶解性铁浓度仅0.2–0.3 nM), 推测固氮生物生长的限制性因子可能为铁[74], 但是并没有在该区域直接实验证明, 尽管北部湾冬季湾口的现场加富培养实验表明生物固氮作用可能受到铁限制[152], 但是在南海北部区域基于痕量金属洁净技术采样发现南海北部区域铁较为充足(0.50 nM), 可能不会限制浮游生物的生长[153], 但是固氮生物生长对铁的需求其实要高于其他浮游植物[71], 并不足以说明铁不是限制因子。粤西琼东上升流区域的研究则认为, 上升流带来大量铁, 而磷却被浮游植物快速利用而缺乏, 最终该区域表现为磷限制[85]。

南海不同海域固氮作用对初级生产力所需氮的贡献有很大差别, 如越南沿岸区域固氮可以支持初级生产力氮需求的0.1—8.2%[18, 154], 而湄公河河口区域贡献率可达47%[61], 三亚湾束毛藻固氮量对初级生产氮贡献率为0.03—1.63%[134], 而粤西琼东上升流海区贡献率仅为0.01—2.52%[85], 南海北部海盆区域暖涡影响区域该贡献率最高可以达到9%[109]。

5 结论与展望

海洋生物固氮研究的发展与研究技术方法密切相关, 技术的突破是研究突破的基础, 如从1961年束毛藻发现到1989年PCR技术应用于海洋固氮研究领域之间将近30年间, 海洋生物固氮的研究并没有取得里程碑式的进展。然而, 二十世纪九十年代分子生物学和15N2同位素示踪法应用于固氮研究领域, 取得了一些突破性进展。在空间分布方面, 生物固氮的研究从传统上认为寡营养盐的低纬度地区拓展到上升流、河口及海湾等富营养盐海域[18, 29, 31, 34–35, 40, 106, 116–121], 从高温高盐的热带亚热带低纬度区域到高纬度寒冷海域[16, 155–156], 从海洋表层到上千米的深层和沉积物[40, 157]。在对固氮生物多样性的认识方面, 从束毛藻到单细胞固氮蓝藻的发现[15, 32, 34, 38–39, 40, 42, 106, 158–159], 再到发现异养固氮细菌的重要性[6–7, 48–50, 160–161]。这些发现, 拓展了对固氮生物及其固氮的传统认识, 表明以前基于大洋寡营养盐区域束毛藻为主要固氮生物估算的固氮量可能低估了生物固氮在全球海洋生物地球化学循环中的地位[38–44]。

固氮作用与反硝化作用分别是海洋氮最重要的源与汇, 这两个过程决定了海洋氮库的收支平衡[162]。然而, 基于已发表的实测固氮速率的数据显示, 海洋中反硝化速率超过固氮速率, 结合态氮的亏损速率高达近200 Tg N·yr-1[163]。但是, 基于模型和沉积物同位素的结果表明, 全球氮收支处于相对平衡状态[164–165], 这就引起人们重新反思对全球海洋生物固氮的再认识, 是否过去的方法存在低估的情况[166]。首先, 在检测方法上, 过去应用广泛的15N2同位素示踪气泡方法比新的海水添加法低估了固氮速率[13, 21], 如在束毛藻为主要固氮者的海域, 该方法可能低估了62%的固氮速率, 而在单细胞固氮蓝藻、共生固氮蓝藻和异养固氮菌为主导的固氮生物群落结构中, 基于海水添加法的固氮量结果是气泡法的6倍之多[124]。基于气泡法和海水添加法的计算世界大洋固氮速率可以从原先估计的103± 8Tg N·yr-1上升到177±8 Tg N·yr-1[124]。其次, 过去对全球海洋生物固氮量的估计都是基于已发现的大洋区域, 较少涉及到近岸如河口、上升流和海湾等高营养盐区域, 高营养盐区域如河口、上升流和沿岸区域是否具有一些曾被忽视的关键类群？以及适应这种环境的机制是什么？迫切需要用现代分子生物学方法继续探究其机制, 有必要对这些区域的生物固氮进行重新评估和再认识。第三, 尽管发现了异养固氮细菌在某些区域占据优势地位[5, 6, 51–53], 但是其固氮活性至今并没有进行有效的评估, 而且一般都不认为异养固氮细菌对固氮具有重要性[7]。相信随着NanoSIMS技术的应用, 这一问题可能会得到一定程度的认知。同时, 近年来组学技术(基因组、转录组、蛋白质组和代谢组)也逐渐应用到海洋生物固氮领域, 可以从基因和代谢网络水平上了解固氮生物与环境之间适应的内在机制。

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Research progress in marine biological nitrogen fixation

LI Zhihong1, LI Jinyou2, LIU Jiaxing3, 4, *

1. Development Promotion Center of Marine Fishery Science and Technology, Ocean Development Experimental Zone of Wanshan, Zhuhai 519005, China 2. School of Data Science, City University of Hong Kong, Hong Kong, China 3. Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Provincial Key Laboratory of Applied Marine Biology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China 4. Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou), Guangzhou 511458, China

Marine nitrogen fixation plays an important role in the global carbon and nitrogen cycles because it can support the nitrogen required for primary production. The importance of unicellular nitrogen-fixing cyanobacteria and heterotrophic nitrogen-fixing bacteria has gradually been known since the application of molecular biology and15N2isotope tracing in nitrogen fixation study in the 1990s. Firstly, it is suggested that the nitrogen fixation amount estimated based onmay beunderestimated. Secondly, traditional study of marine biological nitrogen fixation is limited to the tropical and subtropical oligotrophic regions, while less attention has been paid to high nutrient regions such as upwelling and estuaries. Therefore, it is necessary to re-evaluate and re-understand the nitrogen fixation in these regions. This article reviewed the recent progress of nitrogen fixation, including biodiversity and distribution of diazotrophs, limiting factors, methods, and existing question. In addition, the latest progress and question of nitrogen fixation in the South China Sea were also reviewed.

marine biological nitrogen fixation; unicellular nitrogen-fixing cyanobacteria; heterotrophic nitrogen-fixing bacteria; molecular biology technology; high nutrient regions; South China Sea

10.14108/j.cnki.1008-8873.2021.05.026

P735

A

1008-8873(2021)05-215-16

2020-02-26;

2020-03-29

科技基础资源调查专项(2018FY100105); 南方海洋科学与工程广东省实验室(广州)人才团队引进重大专项(GML2019ZD0401); 广东省促进经济高质量发展专项资金海洋经济发展项目(GDOE[2019]A32); 国家自然科学基金(41806198, 31971432); 广东省基础与应用基础研究基金(2019A1515010896); 广州市科技计划项目(202102020279)

李志红 (1970—), 女, 湖南双峰县人, 硕士, 从事海洋渔业及生态学方面工作, E-mail: 799491187@qq.com

通信作者:刘甲星, 男, 博士, 助理研究员, 研究方向为海洋生态学, E-mail: ljx2ljx@sisio.ac.cn

李志红, 李劲尤, 刘甲星. 海洋生物固氮研究进展[J]. 生态科学, 2021, 40(5): 215–230.

LI Zhihong, LI Jinyou, LIU Jiaxing. Research progress in marine biological nitrogen fixation[J]. Ecological Science, 2021, 40(5): 215–230.