Endospores in the deep marine biosphere: Distribution, abundance and function

科学钻探已经证明了地球深层微生物生物圈的丰富性，以及它在推动碳和养分循环方面的重要性。这些未被充分研究的大型群落提出的一个主要问题是，它们如何应对逐渐埋葬和相关的能量饥饿。这个谜题的一个重要部分可能存在于极慢生长、休眠和微生物死亡之间的灰色地带；在这种情况下，细菌内生孢子可能起着关键作用。内生孢子是抗性细胞实体：代谢不活跃，休眠，结构独特，具有监测其栖息地的能力，以便在有利条件下恢复生长。到目前为止，在海底生物量普查中，对内生孢子的研究还很少。事实上，DNA染色在多大程度上渗透到孢子内仍然存在争议，因此孢子内可能是不可见的，也可能被包括在被计数的细胞池中。另一种化学方法，针对特定的生物标志物二吡啶酸（DPA），最近产生了海底内孢子丰度的第一个数据，并表明大量的营养细胞伴随着同样多的休眠内孢子。在此观察的基础上，我们提出了一项基于dpa的海底沉积物内孢子浓度调查，使用我们团队参与ODP和IODP探险（例如，ODP Leg 201, IODP exp . 301, 311, 315, 316, 317, 322, 329, 337）和其他专门研究海底生物的探险所获得的大量保存样本档案。我们的目标是解决深层生物圈中内生孢子的生态相关性，并解决2013-2023年IODP科学计划定义的两个全球挑战：#5海底生物群落的起源、组成和全球意义；#6海底生物的极限是什么？我们的研究是由内生孢子是深层生物圈的主要贡献者这一假设驱动的。为了验证这一点，我们将a)在大量存在环境细胞浓度和地球化学数据的深海海底样品中调查基于dpa的内孢子丰度，b)调查细胞计数在多大程度上包括内孢子。虽然我们实验室已经完全建立了DPA分析的分析方法，但要可靠地枚举深层生物圈中的内生孢子，需要将DPA细化为孢子转化因子和沉积物中DPA分配的信息。我们还假设，内生孢子不仅在埋藏的极端条件下存活，而且可能在深的、刺激活动的栖息地定居。为了验证这一点，我们将a)通过实验评估在地质时间尺度上保持活力的沉积内生孢子的比例以及它们可能发芽的条件；b)确认微生物活动受到刺激的沉积层中内生孢子萌发的生态相关性。

Scientific drilling has demonstrated the abundance of Earth's deep microbial biosphere, and its importance in driving carbon and nutrient cycles. A major question presented by these large and understudied communities is how they deal with progressive burial and associated energy starvation. One important piece of this puzzle may reside in the gray area between extremely slow growth, dormancy, and microbial death; bacterial endospores may play a critical role in this context. Endospores are resistant cellular entities: metabolically inactive, dormant, and structurally unique, with the ability to monitor their habitat in order to resume growth under favorable conditions. Endospores have so far been poorly accounted for in censuses of subseafloor biomass. Indeed, it remains controversial to what extent DNA stains penetrate into endospores, which therefore might either have remained invisible or been included in the pool of counted cells. An alternative chemical approach, targeting the specific biomarker dipicolinic acid (DPA), has recently produced the first data of endospore abundance in the subseafloor and suggests that the vast population of vegetative cells is accompanied by an equally large population of dormant endospores. Building on this observation we propose a DPA-based survey of endospore concentration in subseafloor sediments, using our large archive of appropriately preserved samples resulting from our group's participation in ODP and IODP expeditions (e.g., ODP Leg 201, IODP Exps. 301, 311, 315, 316, 317, 322, 329, 337), and other expeditions dedicated to subseafloor life. We aim to tackle the ecological relevance of endospores in the deep biosphere and to address two of the global challenges defined by the IODP Science Plan for 2013-2023: #5 What are the origin, composition, and global significance of subseafloor communities and #6 What are the limits of life in the subseafloor? Our research is driven by the hypothesis that endospores are major and insufficiently accounted for contributors to the deep biosphere. To test this we will a) survey DPA-based endospore abundance in a large set of deep subseafloor samples for which contextual cell concentration and geochemical data exists, and b) investigate to which extent cell counts include endospores. While the analytical method for DPA analysis is fully established in our lab, reliable enumeration of endospores in the deep biosphere requires refinement of DPA to spore conversion factors and information on the partitioning of DPA in the sediment. We also hypothesize that endospores not only survive extreme conditions during burial, but may colonize deep, activity-stimulating habitats. To test this we will a) experimentally evaluate the ratio of sedimentary endospores that remain viable over geological time scales and the conditions under which they may germinate and b) confirm the ecological relevance of endospore germination in sediment layers where microbial activity is stimulated.