Enhancing oil production and harvest by combining the marine alga Nannochloropsis oceanica and the oleaginous fungus Mortierella elongata
提高油脂产量与收获效率：结合海洋藻类小球藻（Nannochloropsis oceanica）与产油真菌（Mortierella elongata）

Abstract  摘要

Background  背景

Although microalgal biofuels have potential advantages over conventional fossil fuels, high production costs limit their application in the market. We developed bio-flocculation and incubation methods for the marine alga, Nannochloropsis oceanica CCMP1779, and the oleaginous fungus, Mortierella elongata AG77, resulting in increased oil productivity.
尽管微藻生物燃料相比传统化石燃料具有潜在优势，但其高生产成本限制了其市场应用。我们针对海洋藻类 Nannochloropsis oceanica CCMP1779 和富油真菌 Mortierella elongata AG77 开发了生物絮凝和培养方法，从而提高了油脂生产率。

Results  结果

By growing separately and then combining the cells, the M. elongata mycelium could efficiently capture N. oceanica due to an intricate cellular interaction between the two species leading to bio-flocculation. Use of a high-salt culture medium induced accumulation of triacylglycerol (TAG) and enhanced the contents of polyunsaturated fatty acids (PUFAs) including arachidonic acid and docosahexaenoic acid in M. elongata. To increase TAG productivity in the alga, we developed an effective, reduced nitrogen-supply regime based on ammonium in environmental photobioreactors. Under optimized conditions, N. oceanica produced high levels of TAG that could be indirectly monitored by following chlorophyll content. Combining N. oceanica and M. elongata to initiate bio-flocculation yielded high levels of TAG and total fatty acids, with ~ 15 and 22% of total dry weight (DW), respectively, as well as high levels of PUFAs. Genetic engineering of N. oceanica for higher TAG content in nutrient-replete medium was accomplished by overexpressing DGTT5, a gene encoding the type II acyl-CoA:diacylglycerol acyltransferase 5. Combined with bio-flocculation, this approach led to increased production of TAG under nutrient-replete conditions (~ 10% of DW) compared to the wild type (~ 6% of DW).
通过分别培养后再将细胞结合，M. elongata 菌丝体能够通过两种物种之间复杂的细胞相互作用高效捕获 N. oceanica，从而实现生物絮凝。使用高盐培养基可诱导三酰基甘油（TAG）的积累，并提高 M. elongata 中多不饱和脂肪酸（PUFAs），包括花生四烯酸和二十二碳六烯酸的含量。为了提高藻类中的 TAG 生产力，我们在环境光生物反应器中开发了一种基于铵的有效低氮供应方案。在优化条件下，N. oceanica 生产出高水平的 TAG，可通过监测叶绿素含量间接检测。将 N. oceanica 和 M. elongata 结合以启动生物絮凝，产生了高水平的 TAG 和总脂肪酸，分别占总干重（DW）的约 15% 和 22%，以及高水平的 PUFA。通过过表达编码 II 型酰基辅酶 A：二酰基甘油酰基转移酶 5 的基因 DGTT5，对 N. oceanica 进行遗传工程改造，在营养充足的培养基中实现了更高的 TAG 含量。结合生物絮凝，这种方法使营养充足条件下的 TAG 产量（~10% 的 DW）相比野生型（~6% 的 DW）有所提高。

Conclusions  结论

The combined use of M. elongata and N. oceanica with available genomes and genetic engineering tools for both species opens up new avenues to improve biofuel productivity and allows for the engineering of polyunsaturated fatty acids.
同时利用具有可用基因组和基因工程工具的 M. elongata 和 N. oceanica，为提高生物燃料生产力开辟了新途径，并使多不饱和脂肪酸的工程化成为可能。

Background  背景

Plant and algal oils are among the most energy-dense naturally occurring compounds that can be used as feedstocks for biofuel products. Microalgae have been considered as promising sustainable feedstock for supplanting fossil fuels since the 1970s. Advantages of microalgae over other biofuel feedstocks include high oil yield, short generation times, low agricultural land requirements, reduced fresh water needs, and reduced greenhouse gas emissions during algal cultivation [1,2,3]. To improve lipid productivity of microalgae, many studies have been conducted in model microalgae such as Chlamydomonas reinhardtii [4, 5], and biotechnologically relevant species such as Nannochloropsis [6,7,8,9]. Nannochloropsis species have become a focus for lipid and biofuel research, because these marine microalgae grow fast in open ponds or photobioreactors, and can be grown in seawater with high yields of lipid—up to 60% of dry weight (DW) [10,11,12]. In addition, Nannochloropsis is enriched in high-value polyunsaturated fatty acids (PUFAs) such as omega-3 eicosapentenoic acid (EPA), and it has a small and compact haploid genome (~ 30 Mbp). In recent years, the genomes of many species and strains of Nannochloropsis including N. gaditana and N. oceanica have been sequenced [13,14,15], and genetic engineering methods have been developed for gene disruption, i.e. CRISPR–Cas9 [8, 16, 17], and to generate overproduction systems for triacylglycerols (TAGs) and other target molecules [18, 19].
植物油和藻类油是自然界中能量密度最高的化合物之一，可用作生物燃料产品的原料。自 20 世纪 70 年代以来，微藻被认为是取代化石燃料的可持续原料，具有很大潜力。与其他生物燃料原料相比，微藻的优点包括高油产量、短生成周期、对农业用地需求低、淡水需求少，以及在培养过程中温室气体排放减少[1, 2, 3]。为了提高微藻的脂质生产力，许多研究集中在模式微藻（如莱茵衣藻，Chlamydomonas reinhardtii）[4, 5]和具有生物技术相关性的种类（如小球藻属，Nannochloropsis）[6, 7, 8, 9]上。小球藻因其在脂质和生物燃料研究中的重要性受到关注，因为这些海洋微藻可以在开放池塘或光生物反应器中快速生长，且可在海水中培养，脂质产量可高达干重（DW）的 60%[10, 11, 12]。此外，小球藻富含高价值的多不饱和脂肪酸（PUFAs），如 omega-3 二十碳五烯酸（EPA），并且其基因组小而紧凑（约 30 Mbp）。近年来，包括 N. gaditana 和 N. oceanica 在内的多种小球藻物种和菌株基因组已被测序[13, 14, 15]，同时已开发出基因编辑方法，如 CRISPR–Cas9 技术[8, 16, 17]，以及用于三酰基甘油（TAGs）和其他目标分子过量生产的系统[18, 19]。

In spite of these apparent advantages, the high cost of microalgal-based fuel production prevents its application in the market [20,21,22]. The major barriers for the cost-effective production of microalgal biofuels include (1) high cost for harvesting microalgae; (2) low oil content and suboptimal composition; (3) high cost of lipid extraction; and (4) impasses in sustainable nutrient supply. Among these barriers, harvesting microalgae is particularly challenging because of the small cell size (typically 2–20 μm) and low density (0.3–5 g L⁻¹) of microalgae, which can account for up to 50% of the total cost of biofuel products [23,24,25]. Traditional harvesting methods include chemical flocculation using multivalent cations such as metal salts and cationic polymers to neutralize the negative charge on the surface of microalgal cell walls, filtration for relatively large algae (> 70 μm), sedimentation/floatation for species that either fall out of suspension or float without sufficient mixing, thermal drying, and centrifugation, which have high cost and energy consumption [24,25,26].
尽管微藻基燃料生产具有明显的优势，但其高昂的生产成本阻碍了其市场化应用【20, 21, 22】。微藻生物燃料经济高效生产的主要障碍包括：(1) 微藻收获成本高；(2) 油脂含量低且成分不理想；(3) 油脂提取成本高；以及 (4) 可持续养分供给方面的瓶颈。其中，微藻的收获尤为具有挑战性，因为微藻的细胞体积很小（通常为 2–20 μm），密度较低（0.3–5 g/L），这部分成本可占生物燃料总生产成本的 50%【23, 24, 25】。传统的收获方法包括利用多价阳离子（如金属盐和阳离子聚合物）进行化学絮凝以中和微藻细胞壁表面的负电荷，过滤相对较大的藻类（>70 μm），对于能自然沉降或在无充分搅拌条件下能悬浮的藻种进行沉降/浮选，以及热干燥和离心分离等方法，这些方法成本和能耗均较高【24, 25, 26】。

Recently, bio-flocculation of microalgae with living materials such as bacteria and fungi has gained interest because of their high efficiency and relatively low energy requirement [23, 27,28,29]. In addition, many bio-flocculants can be cultured with nutrients from industrial wastes [29, 30]. The gram-positive bacterium Solibacillus silvestris can efficiently flocculate N. oceanica DUT01 without the addition of chemical flocculants or high-energy inputs. The bacterial flocculant exhibits no growth effect on the algal cells, and it can be reused for lower cost in harvesting [31]. In addition, some bio-flocculants such as filamentous fungi Aspergillus fumigatus and Mucor circinelloides can accumulate 10–15% lipid per DW which can increase the total lipid yield [30]. However, these filamentous fungi are also human pathogens, making them unsuitable for use as bio-flocculants.
近年来，利用细菌和真菌等活性材料进行微藻生物絮凝引起了人们的兴趣，因为其效率高且能耗相对较低【23, 27, 28, 29】。此外，许多生物絮凝剂可以利用工业废料中的养分进行培养【29, 30】。例如，革兰氏阳性菌 **Solibacillus silvestris** 能在无需添加化学絮凝剂或高能耗投入的情况下高效絮凝 **N. oceanica DUT01**。这种细菌絮凝剂对藻细胞无生长影响，并且可以重复使用，从而降低收获成本【31】。此外，一些生物絮凝剂（如丝状真菌 **Aspergillus fumigatus** 和 **Mucor circinelloides**）的干重中可积累 10–15% 的脂质，从而提高总脂质产量【30】。然而，这些丝状真菌也是人类病原体，因此并不适合作为生物絮凝剂使用。

Here we screened oleaginous fungi with regard to their ability to flocculate N. oceanica CCMP1779, a marine alga with a sequenced genome, rapidly growing molecular engineering tool kit, and the ability to produce high levels of TAG [9, 13, 18]. We discovered that N. oceanica could be efficiently flocculated by M. elongata AG77, a soil fungus that accumulates high levels of TAG and arachidonic acid (ARA), and for which a sequenced genome is available [32]. We provide detailed insights into the physical interaction between N. oceanica and M. elongata AG77 and test approaches for increasing the TAG content in N. oceanica by optimizing growth conditions and genetic engineering approaches in combination with bio-flocculation to harvest algal cells.
在本研究中，我们筛选了产油真菌，评估其絮凝海洋藻类 **N. oceanica CCMP1779** 的能力。**N. oceanica CCMP1779** 是一种具有已测序基因组、快速发展的分子工程工具包，并能高效生产三酰基甘油（TAG）的海洋藻类【9, 13, 18】。我们发现，土壤真菌 **M. elongata AG77** 能高效絮凝 **N. oceanica**。**M. elongata AG77** 不仅积累高水平的 TAG 和花生四烯酸 (ARA)，其基因组也已被测序【32】。我们深入研究了 **N. oceanica** 和 **M. elongata AG77** 之间的物理相互作用，并测试了通过优化生长条件、结合基因工程方法以及生物絮凝收获微藻细胞来提高 **N. oceanica** 中 TAG 含量的策略。

Results  结果

N. oceanica cells are captured by the M. elongata mycelium
N. oceanica 细胞被 M. elongata 菌丝捕获。

Fungi were incubated in potato dextrose broth (PDB). Fungal mycelium (~ 3 times of algal biomass) was added to the N. oceanica culture containing log-phase cells in f/2 medium in shaker flasks. After 6-day co-cultivation with M. elongata, N. oceanica cells aggregated in dense green clumps along the mycelium of the fungus (Fig. 1a). The interaction of N. oceanica with filamentous fungi appeared specific to M. elongata, as it was not observed in co-culture with Morchella americana 3668S (Fig. 1). Differential interference contrast (DIC) light microscopy showed dense numbers of N. oceanica cells attached to the M. elongata mycelium (Fig. 1c); in comparison, mycelium of M. americana hardly captured any algal cells (Fig. 1d). Three Mortierella strains, M. elongata AG77, M. elongata NVP64, and M. gamsii GBAus22 were used to test the flocculation efficiency for harvesting of N. oceanica with M. americana as a negative control. All three Mortierella isolates aggregated ~ 10% of algal cells after 2-h of co-culture and up to ~ 15% after 12 h (Fig. 1e). After 6-day co-cultivation, M. elongata AG77 and NVP64 captured ~ 60% of algal cells M. gamsii GBAus 22 captured ~ 25%. The short period of co-cultivation with fungi did not appear to affect the morphology of the algal cells and did not significantly change their diameter (Fig. 1f).
真菌在马铃薯葡萄糖培养液（PDB）中培养。将真菌菌丝体（约为藻类生物量的 3 倍）加入含有对数生长期细胞的 N. oceanica 培养物中，该培养物使用 f/2 培养基，置于摇瓶中培养。在与 M. elongata 共培养 6 天后，N. oceanica 细胞沿着真菌菌丝聚集成密集的绿色团块（图 1a）。N. oceanica 与丝状真菌的相互作用似乎是 M. elongata 特有的，因为在与 Morchella americana 3668S 共培养时未观察到这种现象（图 1）。差分干涉对比（DIC）光学显微镜显示大量 N. oceanica 细胞附着在 M. elongata 菌丝上（图 1c）；相比之下，M. americana 的菌丝几乎未捕获任何藻类细胞（图 1d）。使用了三个 Mortierella 菌株——M. elongata AG77、M. elongata NVP64 和 M. gamsii GBAus22——测试其对 N. oceanica 的絮凝效率，并以 M. americana 作为阴性对照。所有三个 Mortierella 菌株在共培养 2 小时后聚集了约 10%的藻类细胞，12 小时后聚集率达到约 15%（图 1e）。经过 6 天的共培养，M. elongata AG77 和 NVP64 捕获了约 60%的藻类细胞，而 M. gamsii GBAus22 捕获了约 25%。与真菌短期共培养似乎未对藻类细胞的形态产生影响，且未显著改变其直径（图 1f）。

Fig. 1  图 1

Harvesting Nannochloropsis oceanica by bio-flocculation with Mortierella fungi. a, b Co-culture of N. oceanica (Noc) with M. elongata AG77 (a), or Morchella americana 3668S (b). Fungal mycelium collected from PDB medium was added to a log-phase Noc culture grown in flasks and both were incubated for 6 days in flasks containing f/2 medium. The red arrow indicates green aggregates formed by AG77 mycelium and attached Noc cells. c Noc cells attached to AG77 mycelium as shown by differential interference contrast (DIC) microscopy. d No obvious attachment of Noc cells on the 3668S mycelium. e Bio-flocculation efficiency for harvesting Noc cells by cocultivation with Mortierella elongata AG77, M. elongata NVP64, and M. gamsii GBAus22 in f/2 medium. The bio-flocculation efficiency was determined by the cell density of uncaptured cells compared to that of a no-fungus Noc culture control. Morchella 3668S culture was used as a negative control. The results are the average of five biological replicates and error bars indicate standard deviation. Asterisks indicate significant differences relative to the 2-h co-cultures by paired-sample Student’s t-test (*P ≤ 0.05; **P ≤ 0.01). f Measurement of Noc cell size (diameter) in the Noc culture and alga-fungus co-culture
利用毛霉菌收获海洋小球藻（Nannochloropsis oceanica）的生物絮凝方法。 a, b 将 N. oceanica（Noc）分别与 M. elongata AG77（a）或 Morchella americana 3668S（b）共培养。从 PDB 培养基中收集的真菌菌丝体被加入到对数生长期的 Noc 培养液中，二者在含有 f/2 培养基的烧瓶中共培养 6 天。红色箭头指示由 AG77 菌丝体和附着的 Noc 细胞形成的绿色絮凝物。 c 通过微分干涉对比显微镜（DIC）观察到 Noc 细胞附着在 AG77 菌丝体上。 d 未观察到 Noc 细胞明显附着在 3668S 菌丝体上。 e 在 f/2 培养基中共培养时，通过 M. elongata AG77、M. elongata NVP64 和 M. gamsii GBAus22 收获 Noc 细胞的生物絮凝效率。生物絮凝效率通过与无真菌 Noc 培养对照组中未被捕获的细胞密度进行比较来确定。Morchella 3668S 培养组用作阴性对照。结果为五个生物重复的平均值，误差线表示标准差。星号表示与 2 小时共培养组相比存在显著差异（配对样本 Student’s t 检验，*P ≤ 0.05；**P ≤ 0.01）。 f Noc 培养液和藻-真菌共培养液中 Noc 细胞大小（直径）的测量结果。

Physical interaction between the cell walls of N. oceanica and Mortierella fungi
N. oceanica 和 Mortierella 真菌细胞壁之间的物理相互作用

Scanning electron microscopy (SEM) was performed to investigate the physical interaction between N. oceanica and M. elongata strains AG77 (Fig. 2a) and NVP64 (Fig. 2b). Low-magnification images (Fig. 2, top panels) showed an aggregation of algal cells around the fungal mycelium as seen in the light micrographs (Fig. 1c). Higher-magnification images displayed details of the physical interaction between the alga and fungus (Fig. 2, middle and bottom panels). Similar to the cell–wall structure of N. gaditana [33], N. oceanica has extensions on the outer layer of the cell wall, which are attached to the rugged surface of the fungal hyphae; irregular tube-like structures are formed between the algal and fungal cell walls, which very likely contribute to anchoring the algal cells to the mycelium. The M. americana strain 3668S, which has much thicker hyphae (10–20 μm in diameter) than the M. elongata strains AG77 and NVP64 (< 2 μm), showed no obvious capture of N. oceanica cells (Fig. 2c) or flocculation.
利用扫描电子显微镜（SEM）研究了 N. oceanica 与 M. elongata AG77（图 2a）和 NVP64（图 2b）菌株之间的物理相互作用。低倍放大图像（图 2，上部面板）显示，在光学显微图（图 1c）中观察到的真菌菌丝周围聚集了大量藻细胞。高倍放大图像展示了藻类与真菌之间物理相互作用的细节（图 2，中部和下部面板）。与 N. gaditana 的细胞壁结构相似 [33]，N. oceanica 的细胞壁外层具有延伸结构，这些结构附着在真菌菌丝粗糙的表面上；藻类和真菌细胞壁之间形成了不规则的管状结构，这很可能有助于将藻细胞锚定在菌丝上。而 M. americana 菌株 3668S 的菌丝直径较粗（10–20 μm），远大于 M. elongata AG77 和 NVP64 菌株的菌丝直径（< 2 μm），没有明显捕获 N. oceanica 细胞（图 2c）或絮凝现象。

Fig. 2  图 2

Interaction between N. oceanica and Mortierella mycelium co-cultivated in f/2 medium. Fungal mycelium was added to a log-phase Noc culture grown in a flask and then the mixture was incubated for 6 days in f/2 medium. Scanning electron microscopy was performed to investigate the interaction between N. oceanica (Noc) cells and M. elongata AG77 (a) and NVP64 (b). Noc cells are attached to the fungal mycelium as shown in the top panel. Higher magnification micrographs show that Noc cells have a highly structured cell wall with protrusions, with which they attach to the rough surface of the fungal cell wall. The red arrowheads indicate tube-like structures that connect the algal and fungal cell walls. c Morchella americana 3668S mycelium collected from a Noc-3668S culture after 6-day co-cultivation
N. oceanica 与 Mortierella 菌丝在 f/2 培养基中共培养的相互作用。将真菌菌丝加入到对数生长期的 Noc 培养液（在烧瓶中生长）中，然后在 f/2 培养基中孵育 6 天。通过扫描电子显微镜研究 N. oceanica（Noc）细胞与 M. elongata AG77 (a) 和 NVP64 (b)的相互作用。如顶部图所示，Noc 细胞附着在真菌菌丝上。更高倍率的显微照片显示，Noc 细胞具有高度结构化的细胞壁，并带有突起，这些突起使其附着在真菌粗糙的细胞壁表面上。红色箭头指示连接藻类和真菌细胞壁的管状结构。c 从 Noc-3668S 培养液中提取的 Morchella americana 3668S 菌丝，在共培养 6 天后收集。

Flocculation of N. oceanica with Mortierella fungi increases the yield of TAG and PUFAs
利用毛霉真菌对 N. oceanica 进行絮凝可提高 TAG 和 PUFA 的产量

Mortierella fungi are known to produce high amounts of TAG and PUFAs including ARA [34, 35]. Indeed, numerous lipid droplets were observed in both Mortierella and Morchella fungi tested for algae flocculation (Fig. 3a–d). In contrast, N. oceanica had fewer and smaller lipid droplets when grown in nutrient-sufficient f/2 medium with or without fungi (Fig. 3e–i) in shaker flasks. Lipids were extracted and separated by thin-layer chromatography (TLC), and fatty acid methyl esters were quantified by gas chromatography and flame ionization detection (GC–FID) to determine the lipid and fatty acid composition. As shown in Table 1, M. elongata AG77 and M. gamsii GBAus22 had much higher contents of TAG, ARA, total PUFAs and total fatty acids, but less content of EPA compared to N. oceanica, which affects the final yield of these compounds in the alga-fungus aggregate. N. oceanica TAG is mainly composed of saturated and monounsaturated fatty acids such as C16:0 and C16:1 (Fig. 4a), whereas Mortierella fungi have more PUFAs, especially ARA (Fig. 4b). N. oceanica has more EPA in total lipid than in TAG (Fig. 4a), and the alga-fungus aggregate contains ~ 10% ARA and ~ 7% EPA of total lipid (Fig. 4c).
被孢霉属真菌已知能够产生大量的三酰甘油（TAG）和多不饱和脂肪酸（PUFAs），包括花生四烯酸（ARA）[34, 35]。实际上，在测试用于藻类絮凝的被孢霉属和羊肚菌属真菌中，观察到了许多脂质滴（图 3a–d）。相比之下，在营养充足的 f/2 培养基中，无论是否添加真菌，摇瓶培养的 N. oceanica 的脂质滴数量更少且体积更小（图 3e–i）。通过薄层色谱法（TLC）提取并分离脂质，采用气相色谱与火焰离子化检测（GC–FID）对脂肪酸甲酯进行定量分析，以确定脂质及脂肪酸的组成。如表 1 所示，M. elongata AG77 和 M. gamsii GBAus22 的 TAG、ARA、总 PUFAs 和总脂肪酸含量明显高于 N. oceanica，但 EPA 含量较低，这影响了这些化合物在藻类-真菌聚合体中的最终产量。N. oceanica 的 TAG 主要由饱和脂肪酸和单不饱和脂肪酸组成，如 C16:0 和 C16:1（图 4a），而被孢霉属真菌则含有更多的 PUFAs，特别是 ARA（图 4b）。N. oceanica 的总脂质中 EPA 含量高于其 TAG 中的 EPA 含量（图 4a），而藻类-真菌聚合体的总脂质中约含有 10%的 ARA 和约 7%的 EPA（图 4c）。

Fig. 3  图 3

Mortierella fungi have more oil droplets than N. oceanica in f/2 medium. a–d Confocal micrographs showing lipid droplets in the fungal mycelium grown in flasks containing PDB medium. Green fluorescence indicates lipid droplets stained with BODIPY. e Lipid droplets in log-phase N. oceanica (Noc) cells grown in flasks containing f/2 medium. Red, autofluorescence of Noc chloroplast. f–i Lipid droplets in the algal and fungal cells after 6-day co-cultivation in f/2 medium. Fungal mycelium was added to the log-phase Noc culture for 6-day co-culture in flasks. AG77, M. elongata AG77; NVP64, M. elongata NVP64; GBAus22, M. gamsii GBAus22; 3668S, Morchella americana 3668S
在 f/2 培养基中，Mortierella 菌比 N. oceanica 含有更多的油滴。 a–d 共聚焦显微图显示在含有 PDB 培养基的烧瓶中生长的真菌菌丝体内的脂滴。绿色荧光表示用 BODIPY 染色的脂滴。 e 在含有 f/2 培养基的烧瓶中生长的对数生长期 N. oceanica (Noc) 细胞中的脂滴。红色为 Noc 叶绿体的自发荧光。 f–i 在 f/2 培养基中共培养 6 天后的藻类和真菌细胞中的脂滴。将真菌菌丝体加入到对数生长期的 Noc 细胞中，在烧瓶中共培养 6 天。 AG77，M. elongata AG77；NVP64，M. elongata NVP64；GBAus22，M. gamsii GBAus22；3668S，Morchella americana 3668S。

Table 1 Lipid contents of different strains grown in f/2 medium in shaker flasks (mg g⁻¹ total dry weight)
表 1 不同菌株在 f/2 培养基摇瓶中生长的脂类含量（mg/g 干重）

Fig. 4  图 4

Fatty acid profiling of triacylglycerol (TAG) and total lipid in Mortierella fungi, N. oceanica, and algae–fungi aggregates after 6-day co-cultivation. a Fatty acid assays of triacylglycerol and total lipid in log-phase N. oceanica grown in shaker flask containing f/2 medium. Fatty acids are indicated with number of carbons: number of double bonds. Results are the average of five biological replicates with error bars indicating standard deviations (n = 5). b Fatty acid assays of M. elongata AG77 incubated in f/2 medium. Fungal mycelium was collected from PDB medium and then washed and added to f/2 medium and incubated for 6 days. n = 5. c Fatty acids of the algae–fungi aggregates after 6-day co-cultivation in flasks containing f/2 medium. n = 5
对 Mortierella 真菌、N. oceanica 以及藻类-真菌聚集体在共培养 6 天后的三酰甘油（TAG）和总脂质的脂肪酸分析。 a. 在摇瓶中含有 f/2 培养基的对数生长期 N. oceanica 的三酰甘油和总脂质的脂肪酸分析。脂肪酸以碳原子数:双键数表示。结果为五个生物重复的平均值，误差条表示标准偏差（n = 5）。 b. 在 f/2 培养基中培养的 M. elongata AG77 的脂肪酸分析。真菌菌丝体从 PDB 培养基中收集、清洗后加入 f/2 培养基中培养 6 天。n = 5。 c. 在含有 f/2 培养基的烧瓶中共培养 6 天后的藻类-真菌聚集体的脂肪酸分析。n = 5

Compared to regular PDB fungal growth medium, f/2 medium has a high salt concentration and an elevated pH (= 7.6) and lacks sugar [36]. Thus, M. elongata AG77 and M. gamsii GBAus22 were incubated in different media in shaker flasks to test the impact on lipid metabolism of high pH (PDB medium, pH7.6), high pH and high salinity (f/2 + 1% sugar), and high pH and high salinity with sugar starvation (f/2 medium). These adverse conditions generally increased the TAG and total lipid content of M. elongata AG77 and M. gamsii GBAus22, especially under high salinity condition (PDB pH7.6 compared to f/2 + 1% sugar) (Additional file 1: Table S1). Compared to M. gamsii GBAus22, M. elongata AG77 showed a significant increase in TAG and total lipid under high pH (PDB, from pH 5.3 to 7.6), and a lower increase in total lipid, and slight decrease in TAG, upon sugar starvation (f/2 + 1% sugar compared to f/2) (Additional file 1: Table S1). These adverse conditions reduced the contents of ARA and total PUFAs in M. gamsii GBAus22, while EPA increased upon high pH but decreased under high salinity and sugar starvation (Additional file 1: Table S1). In contrast, M. elongata AG77 had increased contents of ARA and PUFAs in response to sugar starvation but these fatty acids decreased under high pH and high salinity conditions; EPA of M. elongata AG77 was decreased under all stress conditions compared to regular growth condition (Additional file 1: Table S1).
与常规的 PDB 真菌培养基相比，f/2 培养基具有较高的盐浓度和较高的 pH 值（=7.6），且不含糖[36]。因此，将 **M. elongata AG77** 和 **M. gamsii GBAus22** 分别在摇瓶中的不同培养基中培养，以测试高 pH 值（PDB 培养基，pH 7.6）、高 pH 值和高盐度（f/2 + 1%糖）、以及高 pH 值和高盐度条件下的缺糖（f/2 培养基）对脂质代谢的影响。这些不利条件通常会增加 **M. elongata AG77** 和 **M. gamsii GBAus22** 的甘油三酯（TAG）和总脂质含量，尤其是在高盐条件下（PDB pH 7.6 与 f/2 + 1%糖相比）（附加文件 1：表 S1）。与 **M. gamsii GBAus22** 相比，**M. elongata AG77** 在高 pH 条件下（PDB，pH 从 5.3 升至 7.6）表现出 TAG 和总脂质的显著增加，而在缺糖条件下（f/2 + 1%糖与 f/2 相比），总脂质的增加较少，TAG 略有下降（附加文件 1：表 S1）。这些不利条件降低了 **M. gamsii GBAus22** 中 ARA 和总 PUFAs 的含量，而 EPA 在高 pH 条件下增加，但在高盐和缺糖条件下减少（附加文件 1：表 S1）。相比之下，**M. elongata AG77** 在缺糖条件下 ARA 和 PUFAs 含量增加，但在高 pH 和高盐条件下这些脂肪酸减少；**M. elongata AG77** 的 EPA 在所有应激条件下均比常规培养条件减少（附加文件 1：表 S1）。

Increasing TAG content in N. oceanica cells using ammonium as the N source
通过使用铵作为氮源提高 N. oceanica 细胞中的 TAG 含量

It has been reported that TAG is the major compound for transitory carbon storage in N. oceanica cells grown under light/dark cycles [37]. However, the TAG content was relatively low when cells were grown under regular conditions [13, 38]. Indeed, N. oceanica cells produced fewer and smaller lipid droplets than the fungi during incubation in f/2 medium visible in confocal micrographs (Fig. 3). To increase TAG yield in N. oceanica, two approaches were employed: nutrient deprivation and genetic engineering. N deprivation is one of the most efficient ways to promote TAG synthesis in microalgae [2, 7, 39]. Following 120-h N deprivation in shaker flasks, TAG accumulated in N. oceanica accounted for up to ~ 70% of the total lipid fraction (Additional file 2: Figure S1A), which is over 20% of DW (Additional file 2: Figure S1B). The content of TAG quickly increased following N deprivation and decreased following N resupply, indicating that N. oceanica cells are very sensitive to the N supply (Additional file 2: Figure S1). Under laboratory conditions, N deprivation of algal cultures can be performed by centrifugation to pellet the algal cells, followed by washes and resuspension in N-depleted medium. However, this approach is not practical during scale up for industrial purposes.
据报道，在光/暗循环条件下生长的 **N. oceanica** 细胞中，TAG 是瞬时碳储存的主要化合物 [37]。然而，在常规条件下培养时，TAG 含量相对较低 [13, 38]。实际上，在 f/2 培养基中孵育时，与真菌相比，**N. oceanica** 细胞产生的脂质液滴更少且更小，这在共聚焦显微照片中清晰可见（图 3）。为了增加 **N. oceanica** 中的 TAG 产量，采用了两种方法：营养剥夺和基因工程。氮（N）剥夺是促进微藻 TAG 合成最有效的方法之一 [2, 7, 39]。在摇瓶中经过 120 小时的氮剥夺后，**N. oceanica** 中积累的 TAG 占总脂质比例高达约 70%（附加文件 2：图 S1A），超过了干重的 20%（附加文件 2：图 S1B）。TAG 的含量在氮剥夺后迅速增加，而在恢复供氮后迅速减少，表明 **N. oceanica** 细胞对氮供应非常敏感（附加文件 2：图 S1）。在实验室条件下，可以通过离心收集藻细胞，随后用无氮培养基洗涤并重悬，从而实现藻类培养的氮剥夺。然而，这种方法在工业规模化过程中并不实际。

Thus, we developed a limited N supply-culturing method for large-volume cultures to induce TAG accumulation largely without compromising growth and biomass yields. To mimic natural cultivation conditions for N. oceanica, such as an open-pond system, we used for these experiments environmental photobioreactors (ePBRs). We grew the algal cells under varying light conditions (0–2000 μmol photons m⁻² s⁻¹) under long-day (14/10 h light/dark) cycles (Additional file 3: Figure S2), and we sparged with 5% CO₂ at 0.37 L min⁻¹ for 2 min per h at 23 °C primarily to adjust the pH in the reactor vessel as previously described in [40]. Illumination in the ePBR is provided by a high-power white LED light on top of a conical culture vessel (total height of 27 cm) containing 330 mL of algal culture (20 cm in depth), which was designed to simulate pond depths from 5 to 25 cm [40]. We substituted several N sources in the f/2 medium for the incubation of N. oceanica containing set amounts of either ammonium, nitrate, or urea. Compared to nitrate and urea, N. oceanica grew faster in the f/2-NH₄Cl medium (Additional file 4: Figure S3A), consistent with previous observations for Nannochloropsis sp. [41] and Nannochloropsis gaditana [42]. The DW of N. oceanica cells per L was also higher in the f/2-NH₄Cl culture after 7-day incubation in the ePBR (Additional file 4: Figure S3B). Intriguingly, the cells grown in f/2-NH₄Cl medium turned from vivid green to yellow following 7-day incubation once they reached stationary phase (Additional file 3: Figure S2A), indicative of chlorophyll degradation in the algal cells. Lipid analysis by TLC (Fig. 5a) and GC–FID (Fig. 5b) demonstrated TAG accumulation during the period of days from 2 to 8 after the culture reached stationary phase (incubation time S2 to S8), which was inversely correlated with chlorophyll content, while cell density and dry weight remained at similar levels during this period (Additional file 4: Figure S3C, D). Previously, to prevent carbon limitation, NaHCO₃ was added to N. oceanica cultures in shaker flasks [43]. Here using the ePBRs, we noticed that the addition of NaHCO₃ prevented acidification of the cultures, which were sparged with 5% CO₂ (Additional file 5: Figure S4A). N. oceanica cells accumulated more TAG upon acidification in the culture medium without NaHCO₃ supply, especially from S6 to S8, compared to the NaHCO₃ culture (Additional file 5: Figure S4A, B).
因此，我们开发了一种有限氮供应的大容量培养方法，以在不显著影响生长和生物量产量的情况下诱导 TAG 的积累。为了模拟 N. oceanica 的自然培养条件（如开放池塘系统），我们在这些实验中使用了环境光生物反应器（ePBRs）。我们在不同光照条件下（0–2000 μmol photons m⁻² s⁻¹）和长日照周期（14/10 小时光/暗）中培养藻细胞（附加文件 3：图 S2），并以 0.37 L min⁻¹的速率通入 5% CO₂，每小时通气 2 分钟，在 23°C 条件下主要用于调节反应器容器内的 pH 值，如文献[40]所述。ePBR 提供的光源为顶部的高功率白色 LED 灯，锥形培养容器的总高度为 27 cm，容纳 330 mL 藻类培养物（深度为 20 cm），设计用于模拟 5 至 25 cm 的池塘深度[40]。我们在 f/2 培养基中替换了几种氮源，用于培养含有设定量的氨、硝酸盐或尿素的 N. oceanica。与硝酸盐和尿素相比，N. oceanica 在 f/2-NH₄Cl 培养基中生长更快（附加文件 4：图 S3A），这与之前关于 Nannochloropsis sp.[41]和 Nannochloropsis gaditana[42]的观察结果一致。在 ePBR 中培养 7 天后，f/2-NH₄Cl 培养基中 N. oceanica 细胞的干重（DW）也更高（附加文件 4：图 S3B）。有趣的是，在 f/2-NH₄Cl 培养基中培养的细胞在进入稳定期后经过 7 天培养（附加文件 3：图 S2A）从鲜绿色变为黄色，这表明藻细胞中的叶绿素发生降解。通过 TLC（图 5a）和 GC–FID（图 5b）进行的脂质分析表明，从培养达到稳定期后的第 2 天到第 8 天（培养时间 S2 至 S8），TAG 积累与叶绿素含量呈负相关，而此期间的细胞密度和干重保持在相似水平（附加文件 4：图 S3C、D）。此前，为防止碳限制，在摇瓶中培养 N. oceanica 时添加了 NaHCO₃[43]。在使用 ePBRs 的实验中，我们注意到添加 NaHCO₃可以防止培养物酸化，而培养物通入 5% CO₂（附加文件 5：图 S4A）。在没有 NaHCO₃供应的培养基酸化条件下，N. oceanica 细胞在培养的 S6 至 S8 期间积累了更多 TAG，相比之下，NaHCO₃培养基中的 TAG 积累较少（附加文件 5：图 S4A、B）。

Fig. 5  图 5

Chlorophyll as proxy of triacylglycerol accumulation. a Analysis of triacylglycerol (TAG) by thin layer chromatography (TLC). Algal cells were incubated in environmental photobioreactor (ePBR) containing f/2-NH4Cl medium after inoculation to stationary phase and then prolonged incubation. Red arrowheads indicate the TAG bands. S1 to S8, day 1 to 8 after the cells reached stationary phase; control, TAG standard. b Inverse correlation of chlorophyll content and TAG-to-total-lipid ratio following prolonged incubation in the ePBR containing f/2-NH₄Cl medium. TAG and total lipid were subjected to transesterification and the resulting fatty acid methyl esters were quantified by GC–FID. r², correlation coefficient; n = 4
叶绿素作为三酰甘油积累的指标。 a. 使用薄层色谱法（TLC）分析三酰甘油（TAG）。在接种到对数生长期后，藻细胞被培养在含有 f/2-NH4Cl 培养基的环境光生物反应器（ePBR）中，并延长培养时间。红色箭头标示出 TAG 条带。S1 到 S8 为细胞达到对数生长期后第 1 天到第 8 天；对照组为 TAG 标准。 b. 在延长培养期间，叶绿素含量与 TAG 占总脂质的比例在含 f/2-NH4Cl 培养基的 ePBR 中呈现负相关。TAG 和总脂质经过转酯化处理，生成的脂肪酸甲酯通过 GC–FID 进行定量分析。r 为相关系数；n = 4。

Increasing the yields of TAG and total fatty acid in alga-fungus aggregates
提高藻菌聚合体中甘油三酯和总脂肪酸的产量

Nannochloropsis oceanica cells were inoculated to ~ 1 × 10⁶ mL⁻¹ in f/2-NH₄Cl medium in the ePBR and then grown to stationary phase. The cultures were further incubated for 8 days to allow the algal cells accumulate high levels of TAG as described above. Following this prolonged incubation in the ePBR, N. oceanica cells had a high TAG content, as shown by direct quantification (Fig. 5b) and confocal microscopy (Fig. 6a). These high-oil algal cells were then transferred from the ePBR to flasks, and ~ 3 times biomass of fungal mycelium was added. This led to the formation of algal–fungal aggregates during 6 days of co-cultivation in flask cultures. We did not attempt to co-cultivate algae and fungi in the ePBRs to avoid fungal contamination of the ePBRs and aggregation disruption by the constant stir bar motion. Following prolonged incubation in the ePBR, N. oceanica cells showed a high TAG content, as determined by direct quantification (Fig. 5b) and confocal microscopy (Fig. 6a). Following a subsequent co-cultivation of algal and fungal cells for 6 days, the algal–fungal aggregates were then collected by mesh filtration for further microscopy and lipid analysis. Both alga and fungus contained a high number of lipid droplets (Fig. 6b), and were enriched in TAG (~ 15% of DW) and total fatty acid (~ 22% of DW) (Fig. 6c), which was much higher than that for f/2 medium-grown algal cells that produced 6.2% TAG and 16.9% total fatty acid of DW (Table 1) with fungal co-cultivation. The overall oil productivity was increased from ~ 0.25 g L⁻¹ day⁻¹ TAG and ~ 0.67 g L⁻¹ day⁻¹ total fatty acid, when the algae were grown in f/2 medium in shaker flasks followed by co-cultivation with fungal cells, to 0.59 g L⁻¹ day⁻¹ TAG and 0.86 g L⁻¹ day⁻¹ total fatty acid, when algae were grown in f/2-NH₄Cl medium in the ePBR followed by co-cultivation with fungal cells. In addition, the oil from these algal–fungal aggregates contained ~ 10% ARA in TAG and total fatty acid (Fig. 6c).
Nannochloropsis oceanica 细胞接种至约 1 × 10 ⁶ mL ⁻¹ 的 f/2-NH ₄ Cl 培养基中，在 ePBR 中培养至稳定期。随后，培养物继续孵育 8 天，使藻细胞积累高水平的 TAG，如上所述。在 ePBR 中的长期孵育后，N. oceanica 细胞显示出较高的 TAG 含量，通过直接定量（图 5b）和共聚焦显微镜（图 6a）得到了验证。这些高油藻细胞随后从 ePBR 转移到烧瓶中，并添加约 3 倍生物量的真菌菌丝体。在烧瓶培养中共培养 6 天后，形成了藻-真菌聚集体。我们没有尝试在 ePBR 中共培养藻类和真菌，以避免 ePBR 被真菌污染以及搅拌棒持续运动对聚集体的破坏。在 ePBR 的长期孵育后，通过直接定量（图 5b）和共聚焦显微镜（图 6a）确定，N. oceanica 细胞显示出较高的 TAG 含量。经过随后 6 天的藻类和真菌细胞共培养后，藻-真菌聚集体通过网过滤收集，用于进一步的显微镜观察和脂质分析。藻类和真菌均含有大量的脂质滴（图 6b），并富含 TAG（约占干重的 15%）和总脂肪酸（约占干重的 22%）（图 6c），显著高于在 f/2 培养基中培养的藻类细胞（通过与真菌共培养产生 6.2% 的 TAG 和 16.9% 的总脂肪酸，见表 1）。总油产率从藻类在摇瓶中的 f/2 培养基培养后与真菌细胞共培养时的 ~0.25 g L ⁻¹ 天 ⁻¹ TAG 和 ~0.67 g L ⁻¹ 天 ⁻¹ 总脂肪酸，提高到藻类在 ePBR 中的 f/2-NH ₄ Cl 培养基培养后与真菌细胞共培养时的 0.59 g L ⁻¹ 天 ⁻¹ TAG 和 0.86 g L ⁻¹ 天 ⁻¹ 总脂肪酸。此外，这些藻-真菌聚集体中的油脂在 TAG 和总脂肪酸中约含有 10% 的花生四烯酸 (ARA)（图 6c）。

Fig. 6  图 6

Increasing triacylglycerol (TAG) content in N. oceanica using limited ammonium as N source. a N. oceanica (Noc) cells produce large lipid droplets during prolonged incubation in the environmental photobioreactor (ePBR) containing f/2-NH₄Cl medium. Noc cells grow fast in f/2-NH₄Cl medium and suffer from nutrient limitation after 8 days in the stationary phase, when the confocal micrographs were taken. Green fluorescence indicates lipid droplets stained with BODIPY, while red fluorescence represents autofluorescence of Noc chloroplasts. b Lipid droplet staining of M. elongata AG77 and Noc cells after 6 days co-cultivation in shaker flasks following the incubation of the algae in ePBRs. c Fatty acid (FA) analyses of triacylglycerol and total lipid in the alga-fungus aggregate as shown in (b). Biomass ratio of TAG or total FA relative to the total cell dry weight (DW). n = 5
通过使用有限的铵盐作为氮源增加 N. oceanica 中的三酰甘油（TAG）含量。 a. 在含有 f/2-NH ₄ Cl 培养基的环境光生物反应器（ePBR）中，N. oceanica（Noc）细胞在长时间孵育期间产生了大量的脂质滴。Noc 细胞在 f/2-NH ₄ Cl 培养基中生长迅速，并在进入静止期 8 天后因营养限制而受到影响，此时拍摄了共聚焦显微照片。绿色荧光表示用 BODIPY 染色的脂质滴，而红色荧光代表 Noc 叶绿体的自发荧光。 b. 对在 ePBR 中孵育后的藻类与真菌（M. elongata AG77 和 Noc 细胞）在摇瓶中共培养 6 天后的脂质滴进行染色。 c. 对（b）中显示的藻类-真菌聚集体的三酰甘油和总脂质进行脂肪酸（FA）分析。三酰甘油或总脂肪酸相对于总细胞干重（DW）的生物质比例。n = 5

Genetic engineering was also performed to increase TAG content in N. oceanica by overexpressing DGTT5 under the control of a strong elongation factor gene (EF) promoter (Additional file 6: Figure S5). Two strains (DGTT5ox3 and ox6) obtained from nuclear transformation displayed a strong increase in the DGTT5 expression compared to the wild-type and empty-vector control by quantitative RT-PCR (Additional file 7: Figure S6A). The construct generated DGTT5 fused to the cerulean fluorescent protein, and the presence of the cerulean protein in DGTT5ox3 and ox6 was confirmed by confocal microscopy (Additional file 7: Figure S6B). Subsequent BODIPY staining showed that the DGTT5oxs lines had more lipid droplets compared to the wild type under regular growth condition (Additional file 7: Figure S6C), which was further confirmed by quantitative lipid analysis (Additional file 7: Figure S6C). Because the elongation factor promotor was inhibited by N starvation, the DGTToxs cells were not incubated in f/2-NH₄Cl medium but grown in regular f/2 medium, and then log-phase cells of DGTT5oxs were co-cultivated with ~ 3 times biomass of M. elongata AG77, and TAG yields of the algal–fungal aggregates were determined. The DGTT5oxs had increased TAG content (~ 10% of DW) compared to the wild type (~ 6% of DW) (Additional file 7: Figure S6E), which was less productive than the cells cultivated in a low N-medium (~ 15% of DW).
通过基因工程改造，通过在强延伸因子基因（EF）启动子的控制下过表达 **DGTT5**，成功提高了 *N. oceanica* 的三酰基甘油（TAG）含量（附加文件 6：图 S5）。通过核转化获得的两种菌株（DGTT5ox3 和 ox6）相比野生型和空载体对照组，DGTT5 的表达在定量 RT-PCR 检测中显示出显著增加（附加文件 7：图 S6A）。构建体将 DGTT5 与蓝色荧光蛋白（cerulean fluorescent protein）融合，且通过共聚焦显微镜证实了 DGTT5ox3 和 ox6 中存在蓝色荧光蛋白（附加文件 7：图 S6B）。随后通过 BODIPY 染色发现，DGTT5oxs 菌株在常规生长条件下相比野生型具有更多的脂质液滴（附加文件 7：图 S6C），这一结果通过定量脂质分析进一步得到确认（附加文件 7：图 S6C）。由于延伸因子启动子在缺氮条件下受到抑制，DGTT5oxs 细胞未在 f/2-NH ₄ Cl 培养基中培养，而是在常规 f/2 培养基中生长。随后，将处于对数生长期的 DGTT5oxs 细胞与约 3 倍生物量的 *M. elongata AG77* 共培养，测定了藻-真菌聚合体的 TAG 产量。结果表明，DGTT5oxs 菌株的 TAG 含量（约占干重的 10%）较野生型（约占干重的 6%）有所提高（附加文件 7：图 S6E），但其产量低于在低氮培养基中培养的细胞（约占干重的 15%）。

Fatty acid and TAG synthesis pathways in M. elongata AG77
M. elongata AG77 中的脂肪酸和甘油三酯（TAG）合成途径

The genome of N. oceanica CCMP1779 has been sequenced and analyzed for the presence of metabolic pathway genes for PUFA and TAG biosynthesis [13], information used in the genetic engineering for increased EPA content [18]. For Mortierella fungi, nuclear transformation methods were established [44, 45], and the M. elongata AG77 genome has been sequenced and annotated [32], but lipid metabolic pathways have not yet been reconstructed. Thus, we applied the genome browser and BLAST tools from the JGI fungal genome portal MycoCosm to predict fatty acid, PUFA, and TAG synthesis pathways for M. elongata AG77. The fatty acid synthesis pathway (Fig. 7a) was predicted according to gene candidates (Additional file 8: Table S2) and previous reports on eukaryotic fatty acid pathways [46, 47]. M. elongata AG77 has a type-I fatty acid synthase with a similar domain organization as found in yeast (Fig. 7b) [46]. Nine elongases and twelve desaturases were identified within the M. elongata AG77 genome for PUFA synthesis, including a ∆15 fatty acid desaturase (FAD) for EPA synthesis (Fig. 7c, Additional file 8: Table S2). Three DGATs and one PDAT (phospholipid:diacylglycerol acyltransferase) were present in the M. elongata AG77 genome, which is similar to what was reported for M. alpina [47].
N. oceanica CCMP1779 的基因组已被测序并分析其多不饱和脂肪酸（PUFA）和三酰基甘油（TAG）生物合成相关的代谢通路基因 [13]，这些信息被用于通过基因工程提高其 EPA 含量 [18]。对于 Mortierella 属真菌，已经建立了核转化方法 [44, 45]，且 M. elongata AG77 的基因组已被测序和注释 [32]，但脂质代谢通路尚未被重建。因此，我们利用 JGI 真菌基因组门户网站 MycoCosm 的基因组浏览器和 BLAST 工具预测了 M. elongata AG77 的脂肪酸、PUFA 和 TAG 合成通路。脂肪酸合成通路（图 7a）是根据候选基因（附加文件 8：表 S2）以及之前关于真核生物脂肪酸通路的研究报告 [46, 47] 所预测的。M. elongata AG77 拥有与酵母中发现的类似的 I 型脂肪酸合成酶，其结构域组织相似（图 7b）[46]。在 M. elongata AG77 基因组中，鉴定出了 9 种延长酶和 12 种去饱和酶用于 PUFA 合成，包括用于 EPA 合成的∆15 脂肪酸去饱和酶（FAD）（图 7c，附加文件 8：表 S2）。此外，在 M. elongata AG77 基因组中发现了 3 种 DGAT（甘油三酯酰基转移酶）和 1 种 PDAT（磷脂：二酰基甘油酰基转移酶），这与 M. alpina 的报道情况类似 [47]。

Fig. 7  图 7

Predicted fatty acid/lipid pathways in M. elongata AG77. Proteins likely involved in the synthesis of fatty acids (FA), polyunsaturated fatty acids (PUFA), and triacylglycerol (TAG) are identified in the sequenced genome of M. elongata AG77 at the JGI fungal genome portal MycoCosm (Additional file 8: Table S2). a FA synthesis. ACP, acyl carrier protein; AT, acetyltransferase; MPT, malonyl/palmitoyl transferase; ACSL, acyl-CoA synthetase; KS, β-ketoacyl synthase; ER, β-enoyl reductase; DH, dehydratase; KR, β-ketoacyl reductase. b Linear domain organization of fatty acid synthase (FASN) of M. elongata AG77. PPT, phosphopantetheine transferase. c PUFA synthesis. ELOVL, fatty acid elongase; FAD, fatty acid desaturase. Fatty acids are designated by the number of total carbon: the number of double bonds. The position of specific double bonds is indicated either from the carboxyl end (∆) or from the methyl end (ω). d TAG synthesis. ALDH, aldehyde dehydrogenase; ADH, alcohol dehydrogenase; GK, glycerol kinase; GPDH, glycerol-3-phosphate dehydrogenase; GPAT, glycero-3-phosphate acyltransferase; PlsC, 1-acyl-sn-glycerol-3-phosphate acyltransferase; LPIN, phosphatidate phosphatase LPIN; PAP, phosphatidate phosphatase 2; Dgk, diacylglycerol kinase; DGAT, diacylglycerol acyltransferase; PDAT, phospholipid diacylglycerol acyltransferase
预测在 **M. elongata AG77** 中的脂肪酸/脂质代谢途径。通过 JGI 真菌基因组门户网站 **MycoCosm**（附加文件 8：表 S2）对 **M. elongata AG77** 的测序基因组分析，识别出可能参与脂肪酸（FA）、多不饱和脂肪酸（PUFA）以及三酰甘油（TAG）合成的蛋白质。 a. **脂肪酸合成**（FA synthesis）。ACP：酰基载体蛋白；AT：乙酰转移酶；MPT：丙二酰/棕榈酰转移酶；ACSL：酰基辅酶 A 合成酶；KS：β-酮酰合酶；ER：β-烯酰还原酶；DH：脱水酶；KR：β-酮酰还原酶。 b. **M. elongata AG77** 的脂肪酸合酶（FASN）的线性结构域组织。PPT：磷酸泛酰巯基乙胺转移酶。 c. **多不饱和脂肪酸合成**（PUFA synthesis）。ELOVL：脂肪酸延长酶；FAD：脂肪酸去饱和酶。脂肪酸通过碳总数:双键数表示。特定双键的位置以羧基末端（∆）或甲基末端（ω）标注。 d. **三酰甘油合成**（TAG synthesis）。ALDH：醛脱氢酶；ADH：醇脱氢酶；GK：甘油激酶；GPDH：甘油-3-磷酸脱氢酶；GPAT：甘油-3-磷酸酰基转移酶；PlsC：1-酰基-sn-甘油-3-磷酸酰基转移酶；LPIN：磷脂酸磷酸酶 **LPIN**；PAP：磷脂酸磷酸酶 2；Dgk：二酰甘油激酶；DGAT：二酰甘油酰基转移酶；PDAT：磷脂-二酰甘油酰基转移酶。

Discussion  讨论

Microalgal biofuel is a renewable and sustainable energy source that can be a supplement or alternative to fossil fuels [1, 2, 26]. Although microalgal biofuel has many competitive advantages, current high costs associated with algal biofuel production limit its commercialization [20,21,22]. To overcome the major challenges in algal biofuel production, including high costs of harvesting, lipid extraction, and nutrient supply, as well as low oil content in algae, we developed a method to harvest the oleaginous marine alga N. oceanica through bio-flocculation with an oleaginous fungus M. elongata AG77. By optimizing the incubation conditions, we have increased TAG biofuel yields.
微藻生物燃料是一种可再生且可持续的能源，可以作为化石燃料的补充或替代品[1, 2, 26]。尽管微藻生物燃料具有许多竞争优势，但由于生产成本高，目前限制了其商业化[20, 21, 22]。为了克服微藻生物燃料生产中的主要挑战，包括收获成本高、脂质提取困难、营养供给成本高以及藻类油含量低等，我们开发了一种利用产油真菌 M. elongata AG77 通过生物絮凝收获产油海藻 N. oceanica 的方法。通过优化培养条件，我们提高了三酰甘油（TAG）生物燃料的产量。

Bio-flocculation of N. oceanica by Mortierella fungi and physical interactions between the partners
由 Mortierella 真菌对 N. oceanica 的生物絮凝及其合作伙伴间的物理相互作用

Physical interaction and symbioses between fungi and algae naturally occur, such as in lichens, where autotrophic algae co-exist with fungi and develop a mutualistic relationship [48]. In fact, about one-fifth of all the known extant fungal species are symbiotically associated with photobionts such as algae and cyanobacteria in lichenized forms [49]. Taking advantage of known algal–fungal interactions, filamentous fungi have been tested for harvesting microalgae in recent years [23, 29]. However, although some of the tested fungal strains including Aspergillus nomius and A. fumigatus can very efficiently flocculate algae, they are not enriched in oil such that they dilute the algal oil in the resulting biomass [50, 51]. In addition, some fungal strains reported to flocculate algae such as A. fumigatus and M. circinelloides are animal pathogens and can threaten human health [28, 30].
真菌与藻类之间的物理相互作用和共生关系在自然界中普遍存在，例如地衣中，自养型藻类与真菌共存并发展出互利关系[48]。实际上，大约五分之一的已知现存真菌物种以地衣化形式与藻类和蓝藻等光合共生体共生[49]。利用已知的藻类与真菌的相互作用，近年来丝状真菌已被用于收集微藻[23, 29]。然而，尽管一些被测试的真菌菌株（包括黄曲霉和烟曲霉）能够非常高效地絮凝藻类，但它们本身的油含量不高，从而会稀释所得生物量中的藻类油[50, 51]。此外，一些报告中提到能够絮凝藻类的真菌菌株，如烟曲霉和环状毛霉，是动物病原体，可能威胁人类健康[28, 30]。

In this study, we sought to use nonpathogenic oleaginous fungi to test their potential for bio-flocculation of microalgae. Among the screened fungi, M. elongata AG77 can be used to efficiently harvest N. oceanica cells (Fig. 1a). M. elongata AG77, which grows in soil and associates with plant roots [52], produces TAG and PUFAs including ARA and EPA (Table 1). N. oceanica cells were captured by the M. elongata AG77 mycelium. With the addition of ~ threefold algal biomass, M. elongata AG77 can flocculate ~ 65% of algal cells after 6-day co-cultivation in flask cultures and was the most efficient isolate tested (Fig. 1c, e). Other studies have shown high efficiency of algal flocculation by fungi [30, 50, 51]. However, the exact nature of the interaction between algae and fungi remains to be determined. Previous studies have suggested that the fungal cell wall has a positive charge that likely attracts and neutralizes the negatively charged algal cells [30, 50, 51], similar to chemical flocculation methods using metal salts and cationic polymers. Fungal cell walls contain chitin, which has a strong positive charge, and the algal flocculation efficiency was correlated with the chitin content (12–16% of DW) in the five fungal strains tested for harvesting Nannochloropsis sp. [50]. However, zeta potential measurements that represent the surface charges do not necessarily correlate with flocculation efficiency [30, 50], suggesting that the flocculation of algae by fungi is not only dependent on the ionic attraction. In this study, we investigated details of the physical interaction between N. oceanica and fungi with SEM. Micrographs show N. oceanica cells trapped within the mesh-like mycelium of M. elongata AG77 and NVP64 (Fig. 2a, b). High-magnification images (×20,000 to ×250,000) show that N. oceanica has extensions along the outer layer of the cell wall, which appear to attach algal cells to the rugged surface of the fungal cell wall. Similar extension structures have also been observed in N. gaditana cells, which are connected to a thin algaenan layer of the N. gaditana cell wall, based on quick-freeze deep-etch EM and analyses of cell wall compositions [33]. Intriguingly, some fiber-like extensions from the N. oceanica cell walls seem to fuse with the surface of the fungal cell wall and form irregular tube-like structures that appear to anchor the algal cells to the mycelium (Fig. 2a, b). However, the Morchella strain 3668S that cannot flocculate N. oceanica, is also not able to capture N. oceanica cells in its mycelium (Fig. 2c). Considering that both Mortierella and Morchella fungi have high chitin contents in their cell wall [53, 54], M. elongata AG77 and NVP64 and M. americana 3668S likely have positive surface charges. A major physical difference between the strains is that M. americana 3668S has much thicker hyphae than the M. elongata strains, suggesting that the size and structure of the mycelium may be important for the physical attraction of algal cells, but fungal exudates and chemotaxis could also underlie efficient bio-flocculation.
在本研究中，我们尝试使用非致病性高油脂真菌测试其对微藻的生物絮凝潜力。在筛选的真菌中，**M. elongata AG77** 能有效收集 **N. oceanica** 细胞（图 1a）。**M. elongata AG77** 生长于土壤中，与植物根系共生 [52]，能够产生甘油三酯（TAG）和多不饱和脂肪酸（PUFAs），包括花生四烯酸（ARA）和二十碳五烯酸（EPA）（表 1）。**N. oceanica** 细胞被 **M. elongata AG77** 的菌丝捕获。加入约三倍于藻类生物量的真菌菌丝后，**M. elongata AG77** 在培养瓶中共培养 6 天，可絮凝约 65%的藻类细胞，是测试中效率最高的菌株（图 1c、e）。其他研究也表明真菌对藻类的絮凝效率较高 [30, 50, 51]。然而，藻类与真菌之间相互作用的具体机制仍需进一步研究。 此前的研究表明，真菌细胞壁带正电荷，可能吸引并中和带负电荷的藻类细胞 [30, 50, 51]，这一机制与使用金属盐和阳离子聚合物的化学絮凝方法类似。真菌细胞壁中含有带强正电荷的几丁质，研究发现，用于收集 **Nannochloropsis** 属藻类的五种真菌菌株，其絮凝效率与几丁质含量（占干重的 12-16%）相关 [50]。然而，表面电荷的ζ电位测量值与絮凝效率并不总是相关 [30, 50]，这表明真菌对藻类的絮凝不仅仅依赖于离子吸引作用。 在本研究中，我们通过扫描电子显微镜（SEM）研究了 **N. oceanica** 与真菌之间的物理相互作用。显微图显示，**N. oceanica** 细胞被 **M. elongata AG77** 和 **NVP64** 的网状菌丝捕获（图 2a、b）。高倍显微图像（放大 20,000 倍至 250,000 倍）显示，**N. oceanica** 的细胞壁外层有延伸结构，这些结构似乎将藻类细胞附着在真菌细胞壁的粗糙表面上。类似的延伸结构也在 **N. gaditana** 细胞中观察到，这些结构连接到 **N. gaditana** 细胞壁的一层薄藻壳层，根据快速冷冻深蚀刻电子显微镜（EM）和细胞壁成分分析得出 [33]。令人感兴趣的是，**N. oceanica** 细胞壁上的一些纤维状延伸结构似乎与真菌细胞壁表面融合，形成不规则的管状结构，将藻类细胞锚定在菌丝上（图 2a、b）。 然而，无法絮凝 **N. oceanica** 的 **Morchella** 菌株 3668S，也无法在其菌丝中捕获 **N. oceanica** 细胞（图 2c）。考虑到 **Mortierella** 和 **Morchella** 真菌的细胞壁中均含有较高含量的几丁质 [53, 54]，**M. elongata AG77**、**NVP64** 和 **M. americana 3668S** 的表面可能都带正电荷。菌株之间的主要物理差异在于，**M. americana 3668S** 的菌丝比 **M. elongata** 菌株的菌丝粗得多，这表明菌丝的大小和结构可能对藻类细胞的物理吸附起重要作用，但真菌分泌物和趋化性也可能是高效生物絮凝的关键因素。

TAG and PUFAs are increased in Mortierella grown in f/2 medium
在 f/2 培养基中生长的毛霉属真菌中，甘油三酯 (TAG) 和多不饱和脂肪酸 (PUFAs) 含量增加

The oleaginous fungi tested in this study produce a large number of lipid droplets, when incubated in regular PDB medium in flaks cultures as shown by confocal microscopy (Fig. 3a–d). To flocculate N. oceanica cells, fungal mycelium was added into the algal culture that contains high concentration of sea salt at pH 7.6 but lacks a sugar supply. The combined stress conditions presented by f/2 medium elevated the TAG and PUFA contents in M. elongata AG77 (Additional file 1: Table S1) and decreased its growth, such that it would not outgrow the algal cells. In fact, N. oceanica cells had normal diameter (Fig. 1f) and low amount of oil when co-cultivated with the fungi (Fig. 3f–i), similar to the cells that were grown alone and those grown in f/2 medium (Fig. 3e).
在本研究中测试的产油真菌在普通 PDB 培养基中培养时，会在瓶式培养中生成大量的脂滴，如共聚焦显微镜所示（图 3a–d）。为了絮凝 **N. oceanica** 细胞，将真菌菌丝体加入含有高浓度海盐但不含糖的藻类培养液中，pH 值为 7.6。由 f/2 培养基提供的联合胁迫条件提高了 **M. elongata AG77** 中 TAG 和 PUFA 的含量（附加文件 1：表 S1），并降低了其生长速度，从而不会超过藻类细胞的生长。事实上，**N. oceanica** 细胞在与真菌共培养时直径正常（图 1f），且油脂含量较低（图 3f–i），与单独培养及在 f/2 培养基中培养的细胞相似（图 3e）。

Mortierella elongata AG77 is a representative strain of Mortierella, a widespread genus of soil fungi, which are industrially important for the production of ARA-rich oil [34, 35]. Mortierella fungi grow fast in nutrient-rich media, and studies have shown that M. elongata CBS 121.71 and M. alpina SD003 can also grow in wastewater [55, 56], while a related species Umbelopsis isabellina can grow on sewage sludge [57], suggesting low-cost incubation methods of growing M. elongata AG77 for the bio-flocculation of algae.
Mortierella elongata AG77 是 Mortierella（一种广泛分布的土壤真菌属）的代表菌株，该属真菌在工业上因生产富含 ARA（花生四烯酸）油而具有重要意义 [34, 35]。Mortierella 真菌在富含营养的培养基中生长迅速，研究表明，M. elongata CBS 121.71 和 M. alpina SD003 也可以在废水中生长 [55, 56]，而与之相关的 Umbelopsis isabellina 物种可以在污水污泥上生长 [57]，这表明通过低成本培养方法培养 M. elongata AG77 用于藻类的生物絮凝是可行的。

Increasing TAG in N. oceanica cells through genetic engineering and by optimizing incubation conditions
通过基因工程和优化培养条件提高 N. oceanica 细胞中的 TAG 含量

In recent years, intensive studies have been performed on Nannochloropsis because of its potential for biofuel production [7, 10,11,12]. Besides its high TAG content, Nannochloropsis is enriched in PUFAs such as EPA, a high-value omega-3 fatty acid [18]. However, Nannochloropsis species, such as N. oceanica, do not have high oil content under favorable growth conditions (Fig. 3e; Additional file 2: Figure S1) [13, 37]. Thus, although the N. oceanica–M. elongata AG77 co-culture generated favorable yields of ARA and EPA, TAG production was only ~ 6% of DW—lower than the ~ 10% TAG yield when using M. elongata AG77 alone (Table 1, Fig. 4). Nannochloropsis accumulates high levels of TAG and total fatty acids in response to stress conditions such as N deprivation (Additional file 2: Figure S1) and following the addition of the ribosome inhibitor cycloheximide [8]. Similarly, TAG and total lipid stimulation by environmental stresses have been commonly observed in the other microalgae such as green algae and diatoms [2, 7]. TAG is usually used as the feedstock for biodiesel and can be converted into high-energy fatty acid methyl esters (FAMEs) by transesterification with glycerol as a valuable byproduct [25, 58]. In addition, TAG is a nonpolar lipid stored in lipid droplets and is more readily extractable with organic solvent than the polar membrane lipids [59]. Thus, the increasing TAG content can simplify the lipid extraction and transesterification, thereby reducing the cost of processing. To increase TAG content in N. oceanica, we developed an N starvation method for N. oceanica using ammonium as the N source. We tested the growth of N. oceanica with three N sources—ammonium, nitrate, and urea—in ePBRs that can mimic natural growth conditions such as open-pond system (Additional file 3: Figure S2) [40]. N. oceanica cells grow significantly faster and have higher biomass in the ammonium medium than the nitrate and urea media, following the same period of incubation (Additional file 4: Figure S3). In addition, N. oceanica cells consumed ammonium faster than nitrate and approached N starvation during the week after the culture reached stationary phase (Fig. 5; Additional file 3: Figure S2A), consistent with the previous observation that Nannochloropsis sp. grow faster in ammonium medium with a significantly higher uptake rate of ammonium than nitrate [41]. N deprivation is one of the most efficient ways to stimulate TAG accumulation in microalgae. However, it is not feasible to centrifuge the cells and change the medium at large industrial scales, as one can do in the laboratory. Considering that N. oceanica cells are very sensitive to the N supply (Additional file 2: Figure S1), feeding ammonium to the culture with limited amount of N, or in multiple-low doses, can allow for rapid cell growth, and provide a means to control the abundance of N in the culture. The N supply can be stopped as the culture reaches the stationary phase such that the cells exhaust their N supply and enter N starvation, while the total cell count and biomass are not compromised (Additional file 4: Figure S3C, D).
近年来，由于小球藻属（Nannochloropsis）在生物燃料生产方面的潜力，人们对其进行了深入研究[7, 10, 11, 12]。除了其高含量的三酰基甘油（TAG），小球藻还富含如 EPA 等高价值的ω-3 多不饱和脂肪酸（PUFAs）[18]。然而，小球藻物种（如 N. oceanica）在良好的生长条件下油脂含量并不高（图 3e；附加文件 2：图 S1）[13, 37]。因此，尽管 N. oceanica 和 M. elongata AG77 的共培养产生了高产量的 ARA 和 EPA，其 TAG 产量仅为细胞干重（DW）的约 6%，低于仅使用 M. elongata AG77 时约 10%的 TAG 产量（表 1，图 4）。小球藻在应激条件下（如氮缺乏，附加文件 2：图 S1）以及添加核糖体抑制剂环己酰亚胺后，会积累高水平的 TAG 和总脂肪酸[8]。类似地，在其他微藻（如绿藻和硅藻）中，通过环境胁迫刺激 TAG 和总脂质的积累也十分常见[2, 7]。 TAG 通常用作生物柴油的原料，通过与甘油转酯化可转化为高能量的脂肪酸甲酯（FAMEs），同时生成有价值的副产品甘油[25, 58]。此外，TAG 是一种储存在脂质小滴中的非极性脂质，比极性膜脂更容易用有机溶剂提取[59]。因此，提高 TAG 含量可以简化脂质提取和转酯化过程，从而降低加工成本。为提高 N. oceanica 中的 TAG 含量，我们开发了一种使用铵盐作为氮源的氮饥饿方法。我们在能够模拟自然生长条件（如露天池塘系统）的 ePBRs 中测试了三种氮源（铵、硝酸盐和尿素）对 N. oceanica 生长的影响（附加文件 3：图 S2）[40]。与硝酸盐和尿素培养基相比，N. oceanica 细胞在铵培养基中生长显著更快，且生物量更高（附加文件 4：图 S3）。此外，N. oceanica 细胞对铵的消耗速度快于硝酸盐，并在培养达到稳定期后一周内接近氮饥饿状态（图 5；附加文件 3：图 S2A），这与之前观察到的小球藻属在铵培养基中生长更快且铵吸收速率显著高于硝酸盐的结果一致[41]。 氮饥饿是刺激微藻中 TAG 积累最有效的方法之一。然而，在工业大规模生产中，无法像实验室那样通过离心细胞并更换培养基。因此，考虑到 N. oceanica 细胞对氮供应非常敏感（附加文件 2：图 S1），可以通过向培养基中分次或限量添加铵来实现快速细胞生长，同时控制培养基中氮的丰度。当培养达到稳定期时停止氮供应，使细胞耗尽氮源并进入氮饥饿状态，同时不影响总细胞数量和生物量（附加文件 4：图 S3C, D）。

We have developed a quick and simple method to indirectly monitor TAG content for N. oceanica using chlorophyll as an indicator. Chlorophyll content is inversely correlated with TAG content during nutrient deprivation (Fig. 5b). In fact, thylakoid membrane degradation, in parallel with TAG accumulation, has been commonly observed in microalgae and plant vegetative tissues. These combined processes can reduce photosynthesis and photooxidative stress, sequester toxic compounds such as free fatty acids, and save the acyl chains for later resynthesis of membranes when conditions improve [7, 39, 60].
我们开发了一种快速简单的方法，通过叶绿素作为指标间接监测 N. oceanica 的 TAG 含量。在营养缺乏期间，叶绿素含量与 TAG 含量呈负相关（图 5b）。实际上，在微藻和植物营养组织中，类囊体膜的降解通常与 TAG 的积累同时发生。这些联合过程可以减少光合作用和光氧化胁迫，隔离游离脂肪酸等有毒化合物，并保存酰基链以备环境条件改善时重新合成膜结构 [7, 39, 60]。

We also noticed that culture acidification caused by CO₂ supply led to faster and higher TAG accumulation in N. oceanica cells (Additional file 5: Figure S4). The described N starvation method should be applicable for most microalgal species, especially for the wild-type and transgenic strains optimized for growth rate, biomass, and stress resistance, but which have low oil content under regular growth condition. Under the right conditions, TAG accumulation occurs once the culture reaches stationary phase/high biomass. The N starvation method we describe will not reduce cell growth or biomass, which has been commonly observed for genetically engineered high TAG strains [8, 9, 19]. On the contrary, the higher the biomass and cell concentration, the faster the algal cells deplete their N source and generate more TAG. Taking advantage of the high TAG-yielding N. oceanica cells produced by the N starvation method (Fig. 6b), we obtained high yields of TAG (~ 15% of DW) and total fatty acids (~ 22% of DW) in the N. oceanica-AG77 aggregate (Fig. 6b, c).
我们还注意到，通过 CO ₂ 供应引起的培养基酸化导致了 N. oceanica 细胞中更快且更高的 TAG 积累（附加文件 5：图 S4）。所描述的氮限制方法应该适用于大多数微藻物种，尤其是那些经过优化以提高生长速率、生物量和抗逆性的野生型和转基因菌株，但在常规生长条件下油含量较低。在适当的条件下，当培养达到稳定期/高生物量时，TAG 积累会发生。我们描述的氮限制方法不会减少细胞生长或生物量，这在基因工程高 TAG 菌株中通常会被观察到[8, 9, 19]。相反，生物量和细胞浓度越高，藻细胞耗尽氮源并产生更多 TAG 的速度越快。利用通过氮限制方法生产的高 TAG 产量的 N. oceanica 细胞（图 6b），我们在 N. oceanica-AG77 聚集体中获得了高产量的 TAG（约占干重的 15%）和总脂肪酸（约占干重的 22%）（图 6b, c）。

Potential of genetic engineering of PUFAs in N. oceanica and M. elongata AG77
N. oceanica 和 M. elongata AG77 中 PUFAs 基因工程的潜力

Both N. oceanica CCMP1779 and M. elongata AG77 generate ARA and EPA: ARA is low in N. oceanica but high in AG77, whereas EPA is high in N. oceanica but low in AG77 (Fig. 4a, b). Besides ARA and EPA, M. elongata AG77 also produces ~ 5% DHA (Fig. 4b). Thus, the complementary production of ARA, EPA, and DHA, high-value products with important health benefits [61], could increase the value of the oil produced by aggregates of N. oceanica and M. elongata AG77. In addition, the genome of N. oceanica CCMP1779 and its BLAST and gene browser tools are available [13], and genetic engineering methods such as CRISPR–Cas9 and overexpression systems have been established for N. oceanica [9, 16, 18]. Recent studies have successfully increased the EPA production by the overexpression of ∆5 and ∆12 FADs in N. oceanica [18] and induced TAG production under normal growth condition by overexpressing N. oceanica DGTT5 [9].
N. oceanica CCMP1779 和 M. elongata AG77 均能生成 ARA 和 EPA：N. oceanica 中的 ARA 含量较低，而 AG77 中的 ARA 含量较高；相反，N. oceanica 中的 EPA 含量较高，而 AG77 中的 EPA 含量较低（图 4a, b）。除了 ARA 和 EPA，M. elongata AG77 还生成约 5%的 DHA（图 4b）。因此，N. oceanica 和 M. elongata AG77 的组合能够互补生产 ARA、EPA 和 DHA，这些高价值产物具有重要的健康益处[61]，从而可以提升其油脂产品的价值。此外，N. oceanica CCMP1779 的基因组及其 BLAST 和基因浏览工具已被开发[13]，并且针对 N. oceanica 的基因工程方法（如 CRISPR-Cas9 和过表达系统）已被建立[9, 16, 18]。最近的研究通过在 N. oceanica 中过表达∆5 和∆12 FADs，成功提高了 EPA 的产量[18]，并通过过表达 N. oceanica DGTT5，在正常生长条件下诱导了 TAG 的生产[9]。

Transformation methods for Mortierella fungi such as M. alpina have been developed [45, 62], and the genome of M. elongata AG77 is available [32]. In this study, we analyzed the genome data and predicted the PUFA and TAG synthesis pathways (Fig. 7), based on the gene candidates involved in the processes (Additional file 8: Table S2), which will facilitate genetic engineering on PUFA and TAG products in M. elongata AG77 in the future.
已经开发了用于 Mortierella 真菌（如 M. alpina）的转化方法[45, 62]，并且 M. elongata AG77 的基因组数据已可获取[32]。在本研究中，我们分析了基因组数据，并基于参与这些过程的基因候选物（附加文件 8：表 S2），预测了 PUFA 和 TAG 的合成途径（图 7）。这将为未来在 M. elongata AG77 中进行 PUFA 和 TAG 产品的基因工程改造提供便利。

Conclusions  结论

Nannochloropsis oceanica CCMP1779 and M. elongata AG77 are model species of algae and fungi, respectively. Both genera contain many other oleaginous strains (e.g., N. gaditana and M. alpina). The bio-flocculation method demonstrated in this study could be performed with other strains leading to synergistic increases in TAG content of the biomass. The N starvation and seawater medium can also be used on transgenic strains with high TAG or high biomass traits, which has the potential to increase oil yield and reduce the cost of oil extraction and transesterification. N. oceanica grows in seawater, and M. elongata AG77 can be incubated with wastewater and sewage that most likely can reduce the cost of nutrient supply. Overall, the combination of N. oceanica and M. elongata AG77 has the potential to overcome some of the barriers for the commercialization of microalgal/fungal biofuels and biomaterials.
海洋小球藻（Nannochloropsis oceanica）CCMP1779 和 AG77 型长形裸头霉（Mortierella elongata）分别是藻类和真菌的模式种。这两个属中还包含许多其他富油菌株（例如，N. gaditana 和 M. alpina）。本研究中展示的生物絮凝方法可以应用于其他菌株，从而协同提高生物质中的三酰甘油（TAG）含量。氮饥饿条件和海水培养基也可以用于具有高 TAG 或高生物质特性的转基因菌株，这有潜力提高油产量并降低油提取和酯交换的成本。N. oceanica 可以在海水中生长，而 AG77 型长形裸头霉可以用废水和污水培养，这很可能降低养分供应的成本。总体而言，N. oceanica 和 AG77 型长形裸头霉的结合有潜力克服微藻/真菌生物燃料和生物材料商业化的一些障碍。

Methods  方法

Materials and growth condition
材料与生长条件

The marine alga Nannochloropsis oceanica CCMP1779 was obtained from the Provasoli-Guillard National Center for Culture of Marine Phytoplankton. N. oceanica DGTT5-overexpressing strains DGTT5ox3 and DGTT5ox6 were generated and examined by quantitative RT-PCR as previously described [9]. The alga was grown in f/2 medium containing 2.5 mM NaNO₃, 0.036 mM NaH₂PO₄, 0.106 mM Na₂SiO₃, 0.012 mM FeCl₃, 0.012 mM Na₂EDTA, 0.039 μM CuSO₄, 0.026 μM Na₂MoO₄, 0.077 μM ZnSO₄, 0.042 μM CoCl₂, 0.91 μM MnCl₂, 0.3 μM thiamine HCl/vitamin B1, 2.05 nM biotin, 0.37 nM cyanocobalamin/vitamin B12 [63], and 20 mM sodium bicarbonate and 15 mM Tris buffer (pH 7.6) to prevent carbon limitation [43]. The cells were grown in batch cultures in two systems: shaker flask with f/2 medium (under ~ 80 μmole photons m⁻² s⁻¹ at 23 °C) or in ePBRs [40] with f/2-NH₄Cl (2.5 mM NH₄Cl replacing 2.5 mM NaNO₃) or f/2-urea (2.5 mM urea replacing 2.5 mM NaNO₃) medium with varying light conditions as indicated (Additional file 3: Figure S2, 0–2000 μmol photons m⁻² s⁻¹ under diurnal 14/10 h light/dark cycle) at 23 °C and sparged with air enriched to 5% CO₂ at 0.37 L min⁻¹ for 2 min per h. For prolonged-incubation in the ePBR, N. oceanica cells were inoculated to ~ 1 × 10⁶ mL⁻¹ in f/2-NH₄Cl medium and grown to stationary phase. The cultures were further incubated for 8 days to increase TAG content.
海洋藻类 Nannochloropsis oceanica CCMP1779 来自 Provasoli-Guillard 国家海洋浮游植物培养中心。通过定量 RT-PCR 方法（如文献[9]所述），构建并检测了 N. oceanica DGTT5 过表达菌株 DGTT5ox3 和 DGTT5ox6。该藻类在 f/2 培养基中培养，培养基包含 2.5 mM NaNO、0.036 mM NaH PO、0.106 mM Na SiO、0.012 mM FeCl、0.012 mM Na EDTA、0.039 μM CuSO、0.026 μM Na MoO、0.077 μM ZnSO、0.042 μM CoCl、0.91 μM MnCl、0.3 μM 盐酸硫胺素/维生素 B1、2.05 nM 生物素、0.37 nM 氰钴胺/维生素 B12[63]，以及 20 mM 碳酸氢钠和 15 mM Tris 缓冲液（pH 7.6）以避免碳限制[43]。细胞分别在以下两种体系中进行批量培养：含 f/2 培养基的摇瓶（在~80 μmol 光子 m s 下、23°C）或 ePBR（含 f/2-NH Cl 培养基（2.5 mM NH Cl 替代 2.5 mM NaNO）或 f/2-尿素培养基（2.5 mM 尿素替代 2.5 mM NaNO），按照指示的不同光照条件（附加文件 3：图 S2，0–2000 μmol 光子 m s，在昼夜 14/10 小时光暗循环条件下）培养，温度为 23°C，并通入富含 5% CO 的空气，每小时以 0.37 L min 的流速通气 2 分钟。在 ePBR 中进行长期培养时，N. oceanica 细胞接种至 f/2-NH Cl 培养基中，初始密度约为 1 × 10 mL，并培养至稳定期。随后将培养物再孵育 8 天以增加 TAG 含量。

Mortierella fungi M. elongata AG77, M. elongata NVP64, and M. gamsii GBAus22 isolates were isolated from soil samples collected in North Carolina (AG77), Michigan (NVP64), USA, and Australia (GBAus22). Morchella americana 3668S was obtained from the USDA NRRL Agriculture Research Station. Fungal samples were incubated in PDB medium (12 g L⁻¹ potato dextrose broth and 1 g L⁻¹ yeast extract, pH5.3) at 23 °C. For the algal–fungal co-cultivation, fungal mycelia were briefly blended into small pieces (~ 1 cm) with a sterilized blender and were collected by centrifugation (3000g for 3 min) after 24-h recovery in PDB medium. The samples were washed twice with f/2 or f/2—NH₄Cl medium and resuspended in 5–10 mL of the respective medium. One-third of the samples were used for determining dry biomass: 1 mL culture was transferred and filtered with pre-dried and -weighed Whatman GF/C filters and dried overnight at 80 °C. The remaining fungal mycelia were added to the N. oceanica culture (~ 3 times to algal biomass) for 6-day co-cultivation on a shaker (~ 60 rpm) under continuous light (~ 80 μmol photons m⁻² s⁻¹) at 23 °C. Cell size and concentration of N. oceanica cultures were determined with a Z2 Coulter Counter (Beckman). The bio-flocculation efficiency of N. oceanica cells using fungal mycelium was determined by the cell density of uncaptured algal cells compared to that of an algal culture control, to which no fungus was added.
从北卡罗来纳州 (AG77)、密歇根州 (NVP64)（美国）以及澳大利亚 (GBAus22) 收集的土壤样本中分离出 Mortierella 真菌 M. elongata AG77、M. elongata NVP64 和 M. gamsii GBAus22 菌株。Morchella americana 3668S 来自美国农业部 NRRL 农业研究站。真菌样本在 PDB 培养基（12 g/L 马铃薯葡萄糖肉汤和 1 g/L 酵母提取物，pH 5.3）中于 23 °C 培养。对于藻类和真菌的共培养，将真菌菌丝用经灭菌的搅拌器短暂搅拌成小段（约 1 cm），并在 PDB 培养基中恢复 24 小时后，通过离心（3000g，3 分钟）收集。样本用 f/2 或 f/2—NH₂Cl 培养基清洗两次，并重悬于 5–10 mL 相应的培养基中。三分之一的样本用于测定干重：取 1 mL 培养液，用预干燥并称重的 Whatman GF/C 滤纸过滤后，在 80 °C 下干燥过夜。剩余的真菌菌丝被加入到 N. oceanica 培养液中（真菌生物量约为藻类生物量的 3 倍），在振荡器上（约 60 rpm）于持续光照（约 80 μmol 光子 m⁻³s⁻⁴）和 23 °C 条件下共培养 6 天。N. oceanica 培养液的细胞大小和浓度通过 Z2 Coulter Counter（Beckman）测定。使用真菌菌丝对 N. oceanica 细胞进行的生物絮凝效率通过未捕获藻细胞的密度与未加入真菌的藻类培养液对照组的密度进行比较来确定。

Light microscopy  光学显微镜

Interactions between the algal and fungal cells were examined by light microscopy using an inverted microscope with DIC function (DMi8, Leica). DIC images were taken of the algae–fungi aggregates after 6-day co-cultivation.
藻类细胞与真菌细胞之间的相互作用通过倒置显微镜（Leica DMi8）搭载微分干涉对比（DIC）功能进行光学显微观察。DIC 图像是在共同培养 6 天后拍摄的藻类-真菌聚集体。

Scanning electron microscopy
扫描电子显微镜

Scanning electron microscopy was performed to investigate the physical interaction between N. oceanica and fungi at the Center for Advanced Microscopy of Michigan State University (CAM, MSU). Algae–fungi aggregates were collected after 6-day co-culture of the alga N. oceanica with M. elongata (AG77 and NVP64) or M. americana 3668S and were fixed in 4% (v/v) glutaraldehyde solution, followed by drying in a critical point dryer (Model 010, Balzers Union). The samples were then mounted on aluminum stubs with high vacuum carbon tabs (SPI Supplies), and were coated with osmium using a NEOC-AT osmium coater (Meiwafosis). The samples were observed with a JSM-7500F scanning electron microscope (Japan Electron Optics Laboratories).
在密歇根州立大学高级显微中心（CAM, MSU），利用扫描电子显微镜研究了 N. oceanica 与真菌之间的物理相互作用。在将 N. oceanica 与 M. elongata（AG77 和 NVP64）或 M. americana 3668S 共培养 6 天后，收集了藻类-真菌聚集体，并固定在 4%（v/v）的戊二醛溶液中，然后在临界点干燥仪（Balzers Union，型号 010）中干燥。随后，将样品用高真空碳胶带（SPI Supplies）固定在铝制样品台上，并用 NEOC-AT 渗锇仪（Meiwafosis）进行锇涂层。最终，使用 JSM-7500F 扫描电子显微镜（日本电子光学实验室）观察样品。

Confocal microscopy  共聚焦显微镜

Confocal microscopy was carried out to visualize and briefly quantify lipid droplets in the alga and the fungi. The samples were stained with 10 μg mL⁻¹ BODIPY 493/503 (ThermoFisher Scientific) in PBS buffer for ~ 30 min at 23 °C. After two washes with PBS buffer, the samples were observed using an Olympus Spectral FV1000 microscope at CAM, MSU. An argon (488 nm) laser and a solid-state laser (556 nm) were used for BODIPY (emission, 510–530 nm) and chloroplast (emission, 655–755 nm) fluorescence. N. oceanica DGTT5 fused to the cerulean fluorescent protein was overproduced using the EF promotor as previously published [9]. The presence of the fluorescent protein in the DGTT5ox strains was detected by confocal microscopy (emission 420–440 nm) using a LSM 510 Meta Confocal Laser Scanning Microscope (Zeiss).
共聚焦显微镜被用于观察并简单定量藻类和真菌中的脂滴。样品用 10 μg/mL 的 BODIPY 493/503（ThermoFisher Scientific）在 PBS 缓冲液中染色，23°C 下孵育约 30 分钟。然后用 PBS 缓冲液清洗两次，利用奥林巴斯 Spectral FV1000 显微镜（位于密歇根州立大学 CAM 实验室）进行观察。BODIPY 的荧光（发射波长 510–530 nm）和叶绿体荧光（发射波长 655–755 nm）分别使用氩激光（488 nm）和固态激光（556 nm）激发。通过 EF 启动子（根据之前发表的研究[9]）过表达了与蓝色荧光蛋白融合的 N. oceanica DGTT5。通过共聚焦显微镜（发射波长 420–440 nm）使用 LSM 510 Meta 共聚焦激光扫描显微镜（Zeiss）检测 DGTT5ox 菌株中的荧光蛋白的存在。

Lipid extraction and analysis
脂质提取与分析

For lipid extraction, log-phase N. oceanica cells grown in f/2 medium were collected by centrifugation (4000g for 5 min). To test lipid content in different media, Mortierella fungi grown in PDB medium were washed twice with different media: PDB medium, pH7.6; f/2 medium with 1% glucose; f/2 medium. The cells were incubated in the respective medium for 48 h and were subsequently collected for lipid extraction by centrifugation (3000g for 3 min). For total lipid extraction, algae–fungi aggregates were collected by mesh filtration and frozen in liquid nitrogen prior to grinding with mortar and pestle. The fine powders were transferred to a pre-weighed and -frozen glass tube, and total lipids were extracted with methanol-chloroform-88% formic acid (1:2:0.1 by volume) on a multi-tube vortexer (1500g for ~ 20 min; Benchmark Scientific), followed by addition of 0.5 volume of 1 M KCl and 0.2 M H₃PO₄. After phase separation by centrifugation (2000g for 3 min), total lipids were collected for TAG separation and fatty acid analysis. The solids were dried at 80 °C overnight to provide the nonlipid biomass.
为了提取脂质，将生长在 f/2 培养基中的对数生长期 N. oceanica 细胞通过离心（4000g，5 分钟）收集。为了测试不同培养基中的脂质含量，将生长在 PDB 培养基中的 Mortierella 真菌用以下不同培养基清洗两次：pH 7.6 的 PDB 培养基；含 1% 葡萄糖的 f/2 培养基；f/2 培养基。细胞在相应的培养基中孵育 48 小时后，通过离心（3000g，3 分钟）收集以提取脂质。 在总脂质提取过程中，通过网状过滤收集藻类和真菌混合体，并在液氮中冷冻后用研钵和研杵研磨成粉末。将细粉转移到预先称重并冷冻的玻璃管中，并用甲醇-氯仿-88% 甲酸（体积比 1:2:0.1）在多管涡旋混合器上进行脂质提取（1500g，约 20 分钟；Benchmark Scientific）。随后加入 0.5 体积的 1 M KCl 和 0.2 M H₃PO₄。经过离心（2000g，3 分钟）进行相分离后，收集总脂质用于 TAG 分离和脂肪酸分析。固体在 80°C 下干燥过夜，得到非脂质生物质。

TAG was separated by TLC using G60 silica gel TLC plates (Machery-Nagel) developed with petroleum ether-diethyl ether-acetic acid (80:20:1 by volume). An internal standard of 5 μg of tridecanoic acid (C13:0) or pentadecanoic acid (C15:0) was added to each tube containing TAG or total lipid. FAMEs were then prepared with 1 M methanolic HCl at 80 °C for 25 min, and were phase separated with hexane and 0.9% NaCl and nitrogen-dried and resuspended in ~ 50 μL of hexane. Gas chromatography and flame ionization detection (Agilent) were used to quantify the FAMEs in TAG and total lipid as previously described [64]. Dry weight of algae–fungi biomass was obtained by summing up nonlipid and total lipid mass.
TAG 使用 G60 硅胶 TLC 板（Machery-Nagel）通过薄层色谱分离，展开溶剂为石油醚-乙醚-乙酸（按体积比 80:20:1）。在每个含有 TAG 或总脂的试管中加入 5 μg 的三癸酸（C13:0）或五癸酸（C15:0）作为内标。随后使用 1 M 甲醇盐酸在 80 °C 下反应 25 分钟制备 FAMEs，通过正己烷和 0.9% NaCl 进行相分离，用氮气吹干并重溶于约 50 μL 的正己烷中。通过气相色谱和火焰离子化检测器（Agilent）对 TAG 和总脂中的 FAMEs 进行定量分析，方法参考文献[64]。藻类-真菌生物质的干重通过非脂质和总脂质量的总和计算得出。

Chlorophyll measurement  叶绿素测定

Nannochloropsis oceanica cells were collected by centrifugation from 1 mL culture aliquots during prolonged incubation in the ePBRs. Chlorophyll of the pelleted cells was extracted with 900 μL of acetone:DMSO (3:2, v/v) for 20 min with agitation at 23 °C, and measured with an Uvikon 930 spectrophotometer (Kontron) [60].
在 ePBRs 中进行长时间培养期间，从 1 mL 培养液中通过离心收集**Nannochloropsis oceanica**细胞。将沉淀的细胞用 900 μL 丙酮:DMSO（3:2，体积比）在 23 °C 下振荡提取 20 分钟。随后使用 Uvikon 930 分光光度计（Kontron）测定叶绿素含量 [60]。

Generation of N. oceanica DGTT5 overexpression strains

The NoDGTT5 sequence was amplified by PCR using the primers: forward, 5′-ATGACGCCGCAAGCCGACATCACCAGCAAGACGA-3′; reverse, 5′-CTCAATGGACAACGGGCGCGTCTCCCACTCC-3′, followed by gel purification with E.Z.N.A. Gel Extraction Kit (OMEGA Biotek) and ligated into the pnoc ox cerulean hyg vector (Additional file 6: Figure S5A) to obtain the final vector pnoc ox DGTT5 cerulean hyg (Additional file 6: Figure S5B) for nuclear transformation. N. oceanica CCMP1779 cells were transformed by electroporation as previously described [13].

Prediction of fatty acid and TAG pathways

The sequenced genome of M. elongata AG77 [32] was annotated for genes and proteins likely involved in the synthesis of fatty acids, PUFAs, and TAGs using by BLAST searches against KOG and KEGG databases at the JGI fungal genome portal MycoCosm M. elongata AG77 v2.0 and by comparison to previously published annotations of lipid pathways of Mortierella alpina [47].

Accession numbers

Sequence data presented in this article can be found in the genome of N. oceanica CCMP1779 at the JGI database (https://genome.jgi.doe.gov/Nanoce1779/Nanoce1779.home.html). Gene ID of N. oceanica DGTT5, CCMP1779_3915; Accession Number, KY273672. Sequence data of M. elongata AG77 can be found at the JGI fungal genome portal MycoCosm (https://genome.jgi.doe.gov/Morel2/Morel2.home.html). Transcript IDs and protein IDs of the genes are listed in the Additional file 8: Table S2.