Enhancing biomass and lipid productivities of Haematococcus pluvialis for industrial raw materials products
提高雨生红球藻的生物质和脂质生产力，用于工业原材料产品

Abstract  摘要

For biofuels and nutraceuticals, the green microalga Haematococcus pluvialis (Chlorophyceae) is a prospective source of biomass and lipids. This study examined how biomass production and lipid accumulation were affected by temperature (10 °C, 20 °C, and 30 °C) and potassium nitrate (KNO₃) concentrations (0.41 g/L, 0.31 g/L, 0.21 g/L, 0.10 g/L, and 0). The findings showed that the largest biomass (0.665 ± 0.200 g/L) was produced at a potassium nitrate concentration of 0.21 g/L at 20 °C, whereas the highest lipid content (46.31 ± 0.026% dry weight) was produced at a temperature without nitrate. Notably, a balanced result was obtained with a modest nitrate content (0.10 g/L) at 20 °C, yielding significant biomass (0.560 ± 0.136 g/L) and lipids (40.30 ± 0.012% dry weight). These results highlight how crucial it is to optimize cultivation settings in order to increase H. pluvialis's dual productivity, offering important new information for its industrial-scale use. By adjusting growing conditions, this research helps meet the need for renewable resources worldwide by promoting the production of high-value bioproducts and sustainable, commercially viable algae-based biofuels.
对于生物燃料和营养保健品而言，绿色微藻血红球藻（Haematococcus pluvialis，隶属绿藻门）是具有潜力的生物质和脂质来源。本研究探讨了温度（10°C、20°C 和 30°C）和硝酸钾（KNO₃）浓度（0.41 g/L、0.31 g/L、0.21 g/L、0.10 g/L 和 0）对生物质生产和脂质积累的影响。研究结果显示，在 20°C 和硝酸钾浓度为 0.21 g/L 时，生物质产量最高（0.665 ± 0.200 g/L），而在无硝酸盐条件下，脂质含量最高（46.31 ± 0.026% 干重）。值得注意的是，在 20°C 和中等硝酸盐浓度（0.10 g/L）下，获得了平衡的结果，生物质产量（0.560 ± 0.136 g/L）和脂质含量（40.30 ± 0.012% 干重）均较显著。这些结果强调了优化培养条件对提高血红球藻双重产量的重要性，为其工业化应用提供了重要的新信息。通过调节生长条件，本研究有助于满足全球对可再生资源的需求，促进高价值生物产品和可持续、商业化藻类生物燃料的生产。

Introduction  介绍

As sustainable sources of high-value compounds, such as biofuels, nutraceuticals, and cosmetics, microalgae have attracted a lot of interest. The ability of Haematococcus pluvialis (Chlorophyceae) to collect significant lipid content and astaxanthin, a strong antioxidant with uses in aquaculture, medicine, and human health products, makes it stand out among these [4, 8]. A major obstacle still stands in the way of producing H. pluvialis in a way that is both economical and scalable.
作为可持续来源的高价值化合物，例如生物燃料、营养保健品和化妆品，微藻引起了广泛的关注。在这些微藻中，雨生红球藻（Haematococcus pluvialis, Chlorophyceae）因其能够积累大量脂质和虾青素而尤为突出。虾青素是一种强效抗氧化剂，广泛应用于水产养殖、医学以及人类健康产品。然而，要以经济且可扩展的方式生产雨生红球藻仍然面临一个重大障碍。

Previous studies have identified a number of environmental parameters, such as light intensity, salinity, and nutrient availability, that affect H. pluvialis production [17]. Among these, temperature and nitrate concentration are important factors that affect biomass growth and lipid production [1, 11].
以往的研究已经确定了多种影响雨生红球藻生产的环境参数，例如光照强度、盐度和养分供应等[17]。其中，温度和硝酸盐浓度是影响生物量增长和脂质生产的重要因素[1, 11]。

Because it reroutes metabolic pathways towards the formation of triacylglycerol (TAG), frequently at the price of cellular growth, nitrogen deprivation is especially effective at causing lipid accumulation [16]. Conversely, strain and cultivation circumstances determine the ideal temperature ranges, which have a direct impact on cellular metabolism and enzymatic activity [18].
由于氮缺乏会改变代谢途径，促进三酰甘油（TAG）的形成，往往以牺牲细胞生长为代价，因此这种方法在诱导脂质积累方面尤为有效[16]。相反，不同的菌株和培养条件决定了最佳温度范围，而温度直接影响细胞代谢和酶活性[18]。

The significance of examining how temperature and nitrate levels interact with H. pluvialis has been underlined by recent research. Nevertheless, the majority of studies concentrate on individual characteristics, offering little information about how they interact [18]. For example, although it has been demonstrated that low nitrate levels increase lipid content, their effect on total biomass yield—a crucial component for industrial applications—is frequently disregarded.
研究表明，温度和硝酸盐水平如何影响**H. pluvialis**（雨生红球藻）之间的相互作用具有重要意义。然而，大多数研究主要集中在单一因素上，对于它们之间的相互作用提供的信息较少 [18]。例如，虽然已有研究表明低硝酸盐水平会增加脂质含量，但它们对总生物量产量（工业应用中至关重要的组成部分）的影响却经常被忽视。

The creation of methods that maximize biomass production and lipid accumulation for commercially feasible uses is constrained by this information gap [5].
这种信息缺口限制了开发能够最大化生物量生产和脂质积累的商业可行方法 [5]。

The combined effects of temperature and nitrate concentrations on H. pluvialis biomass output, lipid productivity, and lipid composition are methodically investigated in this work. Its specific goal is to find growing conditions that optimize lipid content without materially impairing biomass growth. The findings aid in the creation of sustainable and reasonably priced growing techniques for industrial applications by offering a deeper understanding of these variables.
本研究系统地探讨了温度和硝酸盐浓度对雨生红球藻生物量产量、脂质生产力以及脂质组成的综合影响。研究的具体目标是找出能够在不显著影响生物量增长的情况下优化脂质含量的培养条件。研究结果通过深入了解这些变量，为工业应用开发可持续且成本合理的培养技术提供了支持。

Materials and methods  材料与方法

Culture preparation  培养制备

The green microalga Haematococcus pluvialis (Chlorophyceae) UTEX 2505 was obtained from a certified algal culture collection (UTEX, USA) to ensure authenticity and reproducibility. Pre-cultures were maintained in Optimal Haematococcus Medium (OHM) containing macro- and micronutrients necessary for growth (Fabregas et al., 2000). The pre-cultures were incubated under standard laboratory conditions (20 °C, light intensity of 3750 μmol photons m⁻².s⁻¹, or approximatelly 20,490 Lux) for one week before experimental setup to achieve exponential phase growth.
从认证的藻类培养物收藏库（UTEX，美国）获取了绿色微藻雨生红球藻（Haematococcus pluvialis，Chlorophyceae）UTEX 2505，以确保其真实性和可重复性。预培养在含有生长所需宏量和微量营养物质的优化雨生红球藻培养基（OHM）中进行维护（Fabregas 等，2000）。预培养在标准实验室条件下（20°C，光照强度为 3750 μmol photons m⁻²·s⁻¹，或约 20,490 Lux）孵育一周，以达到指数生长期后进行实验设置。

Experimental setup  **实验设置**

Sterilizations of the freshwater and the culture media were maintained by autoclaving at 120 °C and 20 psi for 40 min (NUVE, Model OT012) before the experimental setups in order to limit the contamination with bacteria, protozoa or another species of algae for keeping monospecific cultures of H. pluvialis. Experiments were conducted in 500 mL Erlenmeyer flasks, each containing 300 mL of aut Claved freshwater enriched with culture medium (OHM). Cultures were aerated with air at 0.5 L.min⁻¹ using a precision air pump to ensure uniform mixing and to supply CO₂ at a rate of 2% of the volume of air every hour for a minute as a carbon source.
在实验设置之前，通过在 120 °C、20 psi 条件下高压灭菌 40 分钟（NUVE, Model OT012）对淡水和培养基进行灭菌，以避免细菌、原生动物或其他藻类的污染，从而保持雨生红球藻（**H. pluvialis**）的单一培养。实验在 500 mL 锥形瓶中进行，每个瓶中含有 300 mL 经过高压灭菌的淡水，并补充了培养基（**OHM**）。培养物通过精密气泵以 0.5 L/min 的速率通入空气进行曝气，以确保均匀混合，并每小时通入 2%的空气体积的二氧化碳（**CO₂**）作为碳源，持续 1 分钟。

Illumination was provided by cool white fluorescent lamps at a constant intensity of 3750 μmol photons m⁻² s⁻¹ (or, approximatelly 20,490 Lux), with a 12:12 light/dark cycle to mimic natural conditions. The pH of the medium was monitored daily and adjusted to 7.5 ± 0.2 using sterile 0.1 M NaOH or HCl to maintain metabolic stability.
照明由冷白荧光灯提供，光强恒定为 3750 μmol photons m⁻² s⁻¹（约合 20,490 勒克斯），并采用 12 小时光/12 小时暗的光周期来模拟自然条件。培养基的 pH 值每日监测，并使用无菌 0.1 M NaOH 或 HCl 调整至 7.5 ± 0.2，以维持代谢稳定性。

Optical density and biomass estimation
光密度与生物量估算

Optical density (OD) was measured spectrophotometrically at 680 nm using a UV–Vis spectrophotometer. The wavelength of 680 nm was chosen as it coincides with the absorption peak of chlorophyll a, the primary pigment in H. pluvialis, providing an accurate proxy for cell density [4, 14].
光密度（OD）在 680 nm 处通过紫外-可见分光光度计（UV-Vis spectrophotometer）进行分光光度测量。选择 680 nm 这一波长是因为它与叶绿素 a 的吸收峰重合，而叶绿素 a 是雨生红球藻（*Haematococcus pluvialis*）中的主要色素，这为细胞密度的准确估算提供了可靠的参考依据[4, 14]。

A standard curve correlating OD680 to dry biomass concentration (g/L) was generated by drying known volumes of culture to a constant weight. This calibration allowed for accurate quantification of biomass during the cultivation period. To accurately monitor the growth of Haematococcus pluvialis, both optical density (OD) at 680 nm and dry biomass weight were evaluated during the cultivation period. A significant positive correlation was observed between OD680 and dry weight, with an R² value of 0.95 derived from the calibration curve. This high R² value indicates a strong linear relationship, affirming that optical density measurements can reliably predict biomass content.
通过将已知体积的培养液烘干至恒重，建立了光密度（OD680）与干生物量浓度（g/L）之间的标准曲线。该校准曲线使得在培养期间能够准确量化生物量。为了精确监测雨生红球藻的生长情况，在培养期间同时评估了 680 nm 处的光密度（OD）和干生物量重量。结果显示 OD680 与干重之间存在显著的正相关关系，校准曲线得出的相关系数（R ² ）为 0.95。这个较高的 R ² 值表明两者之间具有较强的线性关系，进一步验证了光密度测量可以可靠地预测生物量含量。

The use of OD680 as a proxy for biomass estimation is supported by its alignment with the absorption peak of chlorophyll a, the predominant pigment in H. pluvialis. By generating a standard curve from dried culture samples, the study validated that absorbance readings at 680 nm provide a robust and reproducible method for tracking culture growth over time. This approach minimizes the need for frequent destructive sampling, facilitating more efficient monitoring in both laboratory and potential industrial-scale operations.
使用 OD680 作为生物量估算的指标得到了支持，因为它与雨生红球藻主要色素叶绿素 a 的吸收峰一致。通过对干燥的培养样品生成标准曲线，该研究验证了 680 nm 处的吸光度读数能够提供一种稳健且可重复的方法来跟踪培养物的生长。该方法减少了频繁破坏性取样的需求，从而在实验室和潜在的工业规模操作中实现更高效的监测。

These findings underscore the utility of spectrophotometric methods in algal biomass research, where precise and non-invasive monitoring tools are essential for optimizing productivity. The strong correlation between optical density and biomass provides a reliable framework for ongoing experiments and real-time monitoring of H. pluvialis under varying environmental and nutrient conditions.
这些研究结果强调了分光光度法在藻类生物量研究中的实用性，在优化生产力过程中，精确且非侵入性的监测工具至关重要。光密度与生物量之间的高度相关性为在不同环境和营养条件下对雨生红球藻的持续实验和实时监测提供了可靠的基础。

Experimental treatment groups
实验治疗组

Potassium nitrate (KNO₃) was used as the primary nitrogen source at five experimental nitrogen concentrations: 0.41 g/L, 0.31 g/L, 0.21 g/L, 0.10 g/L, and 0 g/L. These concentrations were selected to examine the transition from nitrogen-replete to nitrogen-depleted conditions, a key factor in modulating lipid accumulation and biomass production [1, 11].
硝酸钾（KNO₃）被用作主要的氮源，实验设置了五种氮浓度：0.41 g/L、0.31 g/L、0.21 g/L、0.10 g/L 和 0 g/L。这些浓度用于研究从氮充足到氮缺乏条件的转变，这是调控脂质积累和生物质产量的关键因素 [1, 11]。

The experimental temperature treatments involved three temperature conditions:
实验的温度处理包含三种温度条件：

• 10 °C: Suboptimal, simulating cold stress by using temperature-controlled glass chamber (Ugur, Model USS980)
  10 °C：亚理想状态，通过使用温控玻璃箱（Ugur，型号 USS980）模拟冷胁迫

• 20 °C: Optimal for H. pluvialis growth, based on literature [18] by using AC (Air-Conditioner) Unit (Mitshubishi, Industrial Type).
  20 °C：根据文献 [18]，使用空调装置（三菱工业类型）是雨生红球藻生长的最佳温度。

• 30 °C: Elevated, simulating heat stress conditions by using heater-controlled water bath (Memmert WNB-14 + L1 model)
  30 °C：通过使用带加热控制的水浴（Memmert WNB-14 + L1 型号）模拟热应激条件

Lipid Extraction  **脂质提取**

After 10 days of cultivation, cells were harvested by centrifugation at 5000 rpm for 10 min at 4 °C to prevent degradation of lipids. The biomass was washed twice with deionized water to remove any residual salts or impurities and then freeze-dried using a lyophilizer. Freeze-dried biomass was stored at −20 °C until further analysis.
经过 10 天的培养后，通过以 5000 rpm、4 °C 离心 10 分钟收获细胞，以防止脂质降解。将生物质用去离子水清洗两次以去除残留盐分或杂质，然后使用冷冻干燥机进行冻干处理。冻干后的生物质被储存在−20 °C，直至进一步分析。

Total Lipid extraction followed the method described by Bligh and Dyer (1959). Moisture content of the samples was determined by drying overnight at 105 ºC in a laboratory oven (Memmert, Model UN260) and calculating the weight difference. In order to extract and purify all lipids from H. pluvialis, chloroform–methanol (1:2) was added to the algae sample tubes at an amount equal to 3.75 times their water content, and mixed well. The mixture was kept for 30 min at room temperature and centrifuged at 2500 rpm for 10 min in a refrigerated centrifuge (Sorvall RC-5B). The supernatant, containing the lipid, was removed and availability is a critical factor influencing the metabolic shift toward lipid synthesis [11].
总脂质提取采用 Bligh 和 Dyer（1959）描述的方法。样品的水分含量通过在 105 ºC 的实验室烘箱（Memmert，型号 UN260）中过夜干燥并计算重量差异来确定。为了从**H. pluvialis**（雨生红球藻）中提取和纯化所有脂质，向藻类样品管中加入等于其水分含量 3.75 倍的氯仿-甲醇（1:2）混合液，并充分混合。混合物在室温下静置 30 分钟，然后在冷冻离心机（Sorvall RC-5B）中以 2500 rpm 离心 10 分钟。取出含脂质的上清液，并指出可利用性是影响脂质合成代谢转变的关键因素 [11]。

Choice of temperatures  温度选择

The temperature treatments represent realistic environmental and stress conditions that H. pluvialis might encounter during industrial cultivation. Optimal growth (20 °C) was compared to stress-induced conditions (10 °C and 30 °C) to understand their effects on lipid accumulation and biomass yield [5, 18].
温度处理代表了雨生红球藻（*H. pluvialis*）在工业培养过程中可能遇到的真实环境和胁迫条件。研究将其最佳生长温度（20 °C）与胁迫条件（10 °C 和 30 °C）进行比较，以探讨这些条件对脂质积累和生物量产量的影响 [5, 18]。

Spectrophotometric measurement at 680 nm
在 680 nm 波长下的光谱测量

The 680 nm wavelength corresponds to chlorophyll a’s absorbance peak, which is directly proportional to cell concentration in microalgal cultures. This method is widely used due to its simplicity, reliability, and minimal sample processing requirements [4, 24].
680 nm 的波长对应于叶绿素 a 的吸收峰，该吸收峰与微藻培养物中的细胞浓度成正比。由于该方法简单、可靠且对样品的处理要求极低，因此被广泛应用 [4, 24]。

Results  结果

H.pluvialis culture at different tempratures
雨生红球藻在不同温度下的培养

The algae cultivated in the nutritional medium containing 0.21 g/L potassium nitrate showed a greater rise in biomass than the other groups at 10 °C, whereas the group without potassium nitrate showed a lower increase in biomass (Table 1).The group with the least quantity of potassium nitrate in the nutritional medium produced the least amount of lipids, whereas the group without any nitrate produced the highest, according to an analysis of the total lipid content of H. pluvialis cultivated at 10 °C (Table 1).
在含有 0.21 g/L 硝酸钾的营养培养基中培养的藻类在 10°C 时表现出比其他组更高的生物量增长，而不含硝酸钾的组生物量增长较低（表 1）。对在 10°C 条件下培养的雨生红球藻总脂质含量的分析表明，营养培养基中硝酸钾含量最低的组产生的脂质最少，而不含硝酸盐的组产生的脂质最多（表 1）。

Table 1 Optimal haematococcus medium (OHM) used in the cultivation of Haematococcus pluvialis (Fabragas et. al. [12])
表 1 最优雨生红球藻培养基（OHM）用于雨生红球藻的培养（Fabragas 等 [12]）

Table 2 provides the figures for the water quality for each type of algae that was cultivated during the experiment.
表 2 提供了实验中培养的每种藻类的水质数据。

Table 2 Optical density, dry weight and lipid values of H.pluvialis cultured at 10 °C
表 2 10°C 条件下培养的雨生红球藻的光密度、干重和脂质值

The optical densities of H. pluvialis were 0.252 ± 0.054 in the group containing 0.21 g.L⁻¹ KNO₃, 0.194 ± 0.021 in the group containing 0.31 g.L⁻¹ of KNO₃ and 0.193 ± 0.020 in the group containing 0.10 g.L⁻¹ of KNO₃ at 10 °C. The experimental group received 0.41 g.L⁻¹ of KNO₃ had an optical density of 0.187 ± 0.015, and the group with 0 g g.L⁻¹ KNO₃ had 0.176 ± 0.017. The group that contained 0.21 g.L⁻¹ KNO₃ differed from the other groups in a statistically significant way (P < 0.05). The groups that received 0.41 g.L⁻¹ KNO₃, 0.31 g.L⁻¹ KNO₃, and 0.10 g.L⁻¹ KNO₃ did not differ statistically significantly (P > 0.05). The differences between the 0.31 g.L⁻¹ KNO₃, 0.21 g.L⁻¹ KNO₃, and 0.10 g.L⁻¹ KNO₃ groups were found to be statistically significant (P < 0.05), however the difference between the group having no KNO₃ and the group containing 0.41 g.L⁻¹ KNO₃ was not (P < 0.05).
H. pluvialis 的光密度在含有 0.21 g.L⁻¹ KNO₃ 的组中为 0.252 ± 0.054，在含有 0.31 g.L⁻¹ KNO₃ 的组中为 0.194 ± 0.021，在含有 0.10 g.L⁻¹ KNO₃ 的组中为 0.193 ± 0.020（在 10 °C 下）。实验组中加入 0.41 g.L⁻¹ KNO₃ 的光密度为 0.187 ± 0.015，而未加入 KNO₃（0 g.L⁻¹ KNO₃）的组为 0.176 ± 0.017。含有 0.21 g.L⁻¹ KNO₃ 的组与其他组之间存在统计学显著差异（P < 0.05）。含有 0.41 g.L⁻¹ KNO₃、0.31 g.L⁻¹ KNO₃ 和 0.10 g.L⁻¹ KNO₃ 的组之间无统计学显著差异（P > 0.05）。含有 0.31 g.L⁻¹ KNO₃、0.21 g.L⁻¹ KNO₃ 和 0.10 g.L⁻¹ KNO₃ 的组之间的差异具有统计学显著性（P < 0.05），但未添加 KNO₃ 的组与含有 0.41 g.L⁻¹ KNO₃ 的组之间的差异不具有统计学显著性（P > 0.05）。

The dry weight of H. pluvialis was 0.504 ± 0.142 in the treatment group received 0.21 g.L⁻¹ KNO₃, 0.379 ± 0.133 in the treatment group received 0.10 g.L⁻¹ KNO₃, 0.352 ± 0.057 in the treatment group received 0.31 g.L⁻¹ KNO₃ and 0.328 ± 0.046 in the 0.41 g.L⁻¹ KNO₃ group and 0.303 ± 0.045 for group received 0 g.L⁻¹ of KNO₃. Statistically significant differences (P < 0.05) were seen between the treatment groups and the highest dry weight was determined in the group received 0.21 g.L⁻¹ of KNO₃. The experimental group received 0.31 g.L⁻¹ KNO₃ and the 0.10 g.L⁻¹ KNO₃ group did not differ significantly (P < 0.05), nor did the 0 g.L⁻¹ KNO₃-free group and the 0.31 g.L⁻¹ KNO₃ group or the 0.21 g.L⁻¹ KNO₃ group and the 0.10 g.L-1 group.
H. pluvialis 的干重在接受 0.21 g/L KNO₃ 的处理组中为 0.504 ± 0.142，在接受 0.10 g/L KNO₃ 的处理组中为 0.379 ± 0.133，在接受 0.31 g/L KNO₃ 的处理组中为 0.352 ± 0.057，在接受 0.41 g/L KNO₃ 的处理组中为 0.328 ± 0.046，而在未添加 KNO₃（0 g/L）的组中为 0.303 ± 0.045。统计分析显示，处理组之间存在显著差异（P < 0.05），且干重最高的组为接受 0.21 g/L KNO₃ 的组。接受 0.31 g/L KNO₃ 的实验组与 0.10 g/L KNO₃ 的组之间无显著差异（P < 0.05）；同样，未添加 KNO₃ 的组（0 g/L）与 0.31 g/L KNO₃ 的组之间，或 0.21 g/L KNO₃ 的组与 0.10 g/L 的组之间也无显著差异。

The lipid accumulation results at 10 °C were, in order, 41.90 ± 0.015 for the KNO₃-free group, 23.37 ± 0.085 for the 0.10 g.L⁻¹ KNO₃ group, 14.09 ± 0.010 for the 0.21 g.L⁻¹ KNO₃ group, and 13.00 ± 0.010 for the 0.31 g.L⁻¹ KNO₃ group with 5.16 ± 0.039 and 0.41 g.L⁻¹ KNO₃ group. Statistical analysis of the data revealed a substantial (P < 0.05) difference between the groups and was the highest the KNO₃-free group cultivated at 10 °C.
在 10°C 下的脂质积累结果依次为：KNO ₃ -缺乏组 41.90 ± 0.015，0.10 g.L ⁻¹ KNO ₃ 组 23.37 ± 0.085，0.21 g.L ⁻¹ KNO ₃ 组 14.09 ± 0.010，0.31 g.L ⁻¹ KNO ₃ 组 13.00 ± 0.010，以及 5.16 ± 0.039 和 0.41 g.L ⁻¹ KNO ₃ 组。数据的统计分析表明，各组之间存在显著性差异（P < 0.05），其中在 10°C 下培养的 KNO ₃ -缺乏组的脂质积累最高。

Algae grown at 20 °C had their optical densities and dry weights measured daily (Table 3).
在 20°C 下生长的藻类每天测量其光密度和干重（表 3）。

Table 3 Water quality values of trials conducted at 10 °C
表 3 10°C 条件下试验的水质数值

Examining the optical density and dry weight figures reveals that the 0.21 g.L⁻¹ KNO₃ group has the most biomass growth and the zero potassium nitrate group has the lowest biomass increase. Furthermore, orange coloring was seen in the group on the fourth day without potassium nitrate.
通过观察光密度和干重数据可以看出，0.21 g/L KNO₃组的生物质增长最多，而零硝酸钾组的生物质增长最少。此外，在没有硝酸钾的组中，第四天出现了橙色。

Following the nine days of production, the algae were harvested by centrifugation at 4000 rpm for five minutes, and lipid extraction was carried out following a 12-h drying period at 100 °C. Table 4 provides a list of Dry weight, lipid levels, and optical density of H. pluvialis microalgae cultivated at 20 °C.
经过九天的培养后，藻类以 4000 rpm 离心五分钟进行收获，并在 100°C 干燥 12 小时后进行脂质提取。表 4 列出了在 20°C 培养的雨生红球藻的干重、脂质含量和光密度。

Table 4 Optical density, dry weight and lipid values of H.pluvialis microalgae cultured at 20 °C
表 4 20°C 条件下培养的 H. pluvialis 微藻的光密度、干重和脂质值

The experiment's optical density data at 20 °C were analyzed, and they showed that: 0.276 ± 0.062 to 0.31 g.L⁻¹ KNO₃ group, 0.274 ± 0.072 to 0.21 g.L⁻¹ KNO₃ group: 0.10 g.L⁻¹ KNO₃ to 0.244 ± 0.042 group: 0.41 g.L⁻¹ KNO₃ to 0.236 ± 0.051 and the group comprising 0.184 ± 0.019 and 0 g.L⁻¹ KNO₃, respectively. Based on a statistical analysis of the data, there is a significant difference between the groups that contained 0 g.L⁻¹ KNO₃) was discovered (P < 0.05) and the other groups, but not between the 0.31 g.L⁻¹ KNO₃ and 0.21 g.L⁻¹ KNO₃ or between the 0.41 g.L⁻¹ KNO₃ and 0.10 g.L⁻¹ KNO₃ groups (P > 0.05).
在 20 °C 条件下对实验的光密度数据进行分析，结果显示：0.276 ± 0.062 至 0.31 g.L ⁻¹ KNO ₃ 组，0.274 ± 0.072 至 0.21 g.L ⁻¹ KNO ₃ 组；0.10 g.L ⁻¹ KNO ₃ 至 0.244 ± 0.042 组；0.41 g.L ⁻¹ KNO ₃ 至 0.236 ± 0.051 组，以及包含 0.184 ± 0.019 和 0 g.L ⁻¹ KNO ₃ 的组。基于数据的统计分析，发现含有 0 g.L ⁻¹ KNO ₃ 的组与其他组之间存在显著差异（P < 0.05），但 0.31 g.L ⁻¹ KNO ₃ 与 0.21 g.L ⁻¹ KNO ₃ 组之间，或者 0.41 g.L ⁻¹ KNO ₃ 与 0.10 g.L ⁻¹ KNO ₃ 组之间没有显著差异（P > 0.05）。

Upon examination of the dry weight data, we find 0.31 g.L⁻¹ KNO₃ and 0.664 ± 0.200 group, 0.21 g.L⁻¹ KNO₃ and 0.659 ± 0.232 group; 0.370 ± 0.063 with 0 g.L⁻¹ KNO₃ group; and 0.562 ± 0.136 with 0.10 g.L-1 group. When the values were statistically analyzed, the group with 0 g.L⁻¹ KNO₃ was statistically different from all other groups, and there were no significant differences between the 0.31 g.L⁻¹ KNO₃ group and 0.21 g.L⁻¹ KNO₃ group or between the 0.10 g.L⁻¹ KNO₃ group and 0.41 g.L⁻¹ KNO₃ group seen (P < 0.05).
在对干重数据进行检查后，我们发现：0.31 g.L ⁻¹ KNO ₃ 组为 0.664 ± 0.200，0.21 g.L ⁻¹ KNO ₃ 组为 0.659 ± 0.232；0 g.L ⁻¹ KNO ₃ 组为 0.370 ± 0.063；0.10 g.L-1 组为 0.562 ± 0.136。通过统计分析发现，0 g.L ⁻¹ KNO ₃ 组与所有其他组存在显著差异，而 0.31 g.L ⁻¹ KNO ₃ 组与 0.21 g.L ⁻¹ KNO ₃ 组之间，以及 0.10 g.L ⁻¹ KNO ₃ 组与 0.41 g.L ⁻¹ KNO ₃ 组之间没有显著差异（P < 0.05）。

Lipid-related data: 51.31 ± 0.026 for 0 g.L⁻¹ KNO_3; 0.21 g.L⁻¹ KNO₃ is the group with 19.44 ± 0.088, 0,10 g.L⁻¹ KNO₃ 40.30 ± 0.012. As for the group 0.31 g.L⁻¹ KNO₃, 28.31 ± 0.014; 10.84 ± 0.517 for the group 0.41 g.L⁻¹ KNO₃. The statistical analysis revealed that there were significant differences (P < 0.05) among all the groups.
与脂质相关的数据：0 g.L ⁻¹ KNO _3; 组为 51.31 ± 0.026；0.21 g.L ⁻¹ KNO ₃ 组为 19.44 ± 0.088；0.10 g.L ⁻¹ KNO ₃ 组为 40.30 ± 0.012；0.31 g.L ⁻¹ KNO ₃ 组为 28.31 ± 0.014；0.41 g.L ⁻¹ KNO ₃ 组为 10.84 ± 0.517。统计分析表明，所有组之间存在显著差异（P < 0.05）。

Table 5 lists the water quality values for the algae that were grown during the experiment.
表 5 列出了实验中培养藻类的水质数值。

Table 5 Water quality values of trials conducted at 20 °C
表 5 20°C 条件下试验的水质数值

Examining the optical density and dry weight values of the experiments carried out at 30 °C, it is observed that the biomass increased at similar values in the remaining groups, whereas the biomass did not increase much in the group containing 0 g.L⁻¹ KNO₃. It was found that the orange coloring in this experiment began on the third day (Table 6).
在 30°C 下进行的实验中，通过观察光密度和干重值发现，其余组的生物量增长值相似，而含有 0 g/L KNO₃的组中生物量增长不明显。实验中发现橙色染色从第三天开始出现（表 6）。

Table 6 Optical density, Dry weight and Lipid values of H. pluvialis cultured at 30 °C
表 6 H. pluvialis 在 30°C 培养时的光密度、干重和脂质值

At 30 °C, the optical density findings for the 0.21 g.L⁻¹ KNO₃ group were 0.279 ± 0.081, for the 0.10 g.L⁻¹ KNO₃ group it was 0.263 ± 0.067, for the 0.31 g.L⁻¹ KNO₃ group it was 0.248 ± 0.069, and for the 0.41 g.L⁻¹ KNO₃ group it was 0.243 ± 0.061. This group is described as having 0.189 ± 0.022 and 0 g.L⁻¹ KNO₃. Statistical analysis revealed that there were no significant differences (P > 0.05) between the 0.31 g.L⁻¹ KNO₃; 0.21 g.L⁻¹ KNO₃ and 0.10 g.L⁻¹ KNO₃ groups, however there were significant differences (P < 0.05) between the 0.41 g.L⁻¹ KNO₃ group and the group that contained 0 g.L⁻¹ KNO₃ (Table 5).
在 30 °C 时，0.21 g.L ⁻¹ KNO ₃ 组的光密度结果为 0.279 ± 0.081，0.10 g.L ⁻¹ KNO ₃ 组为 0.263 ± 0.067，0.31 g.L ⁻¹ KNO ₃ 组为 0.248 ± 0.069，0.41 g.L ⁻¹ KNO ₃ 组为 0.243 ± 0.061。该组的光密度为 0.189 ± 0.022，且不含 KNO ₃ （0 g.L ⁻¹ KNO ₃ ）。统计分析显示，0.31 g.L ⁻¹ KNO ₃ 、0.21 g.L ⁻¹ KNO ₃ 和 0.10 g.L ⁻¹ KNO ₃ 组之间无显著性差异（P > 0.05），但 0.41 g.L ⁻¹ KNO ₃ 组与不含 KNO ₃ （0 g.L ⁻¹ KNO ₃ ）的组之间存在显著性差异（P < 0.05）（表 5）。

Upon analyzing the dry weight data, the following groups are found: 0.21 g.L⁻¹ KNO₃, 0.608 ± 0.229; 0.10 g.L⁻¹ KNO₃, 0.561 ± 0.190; 0.31 g.L⁻¹ KNO₃, ± 0.295; 0.41 g.L⁻¹ KNO₃, 0.504 ± 0.174; and 0 g.L⁻¹ KNO₃. Upon analyzing the statistical outcomes of the experiment carried out at 30 °C in terms of dry weight, it was determined that there were no significant differences (P > 0.05) between the 0.21 g.L⁻¹ KNO₃ group and the 0.10 g.L⁻¹ KNO₃ group, or between the 0.31 g.L-1 g.L⁻¹ KNO₃ group and the 0.41 g.L⁻¹ KNO₃ group, and between the 0.21 g.L⁻¹ KNO₃ group and the 0.10 g.L⁻¹ KNO₃ group and the 0.41 g.L⁻¹ KNO₃ group. Table 5 shows that the other groups' differences from the contained group were not statistically significant (P < 0.05).
在分析干重数据后，发现以下组别：0.21 g.L ⁻¹ KNO ₃ ，0.608 ± 0.229；0.10 g.L ⁻¹ KNO ₃ ，0.561 ± 0.190；0.31 g.L ⁻¹ KNO ₃ ，± 0.295；0.41 g.L ⁻¹ KNO ₃ ，0.504 ± 0.174；以及 0 g.L ⁻¹ KNO ₃ 。在分析 30°C 条件下实验的统计结果（以干重为指标）后发现，0.21 g.L ⁻¹ KNO ₃ 组与 0.10 g.L ⁻¹ KNO ₃ 组之间，0.31 g.L ⁻¹ KNO ₃ 组与 0.41 g.L ⁻¹ KNO ₃ 组之间，以及 0.21 g.L ⁻¹ KNO ₃ 组与 0.10 g.L ⁻¹ KNO ₃ 组和 0.41 g.L ⁻¹ KNO ₃ 组之间均无显著性差异（P > 0.05）。表 5 显示，其他组与包含组之间的差异在统计学上不显著（P < 0.05）。

Upon analyzing the lipid quantities generated by each group, the 0 g.L⁻¹ KNO₃ group produced 50.14 ± 0.015; 0.10 g.L⁻¹ KNO_3, 30.36 ± 0.010; 0.21 g.L⁻¹ KNO₃ group 21.54 ± 0.014; 0.31 g.L⁻¹ KNO₃, 16.81 ± 0.014; 0.41 g.L⁻¹ KNO₃, 9.56 ± 0.295 it was observed to be listed. Following a statistical analysis, it was determined that there were substantial (P < 0.05) differences between each group.
通过分析每组产生的脂质量，发现 0 g.L ⁻¹ KNO ₃ 组为 50.14 ± 0.015；0.10 g.L ⁻¹ KNO _3, 组为 30.36 ± 0.010；0.21 g.L ⁻¹ KNO ₃ 组为 21.54 ± 0.014；0.31 g.L ⁻¹ KNO ₃ 组为 16.81 ± 0.014；0.41 g.L ⁻¹ KNO ₃ 组为 9.56 ± 0.295。通过统计分析确定，各组之间存在显著性差异（P < 0.05）。

The algae were harvested at 4000 rpm for 5 min and dried at 100 °C for 12 h following a 9-day manufacturing cycle. By extracting the lipids from dried algae, it was noted.
藻类在 4000 rpm 下收获 5 分钟后，于 100°C 下干燥 12 小时，整个过程为期 9 天的生产周期。通过从干燥的藻类中提取脂质，得出了以下观察结果。

Examining the lipid graph for the experiments carried out at 30 °C, it can be observed that the algae cultivated in the zero potassium nitrate nutritional medium had the maximum quantity of lipids, while the first group with the highest nitrate content had the lowest amount (Table 6). Table 7 provides the experiment’s water quality values.
在观察 30°C 条件下进行的实验脂质图时发现，在零硝酸钾营养介质中培养的藻类含有最多的脂质，而硝酸盐含量最高的第一组藻类的脂质含量最低（见表 6）。实验的水质数值见表 7。

Table 7 Water Quality values of trials conducted at 30 °C
表 7 30°C 条件下试验的水质值

Fatty acid composition results
脂肪酸组成结果

By using GC-FID, the fatty acid composition of the oil extracted from H. pluvialis microalgae cultivated in growth conditions with 0.10 g.L⁻¹ KNO₃ and at a water temperature of 20 °C was ascertained. Information on these outcomes can be seen in Table 8.
通过使用气相色谱-氢火焰离子化检测器（GC-FID），测定了在含有 0.10 g/L KNO₃的培养条件和水温为 20°C 下培养的雨生红球藻（H. pluvialis）微藻所提取油脂的脂肪酸组成。相关结果见表 8。

Table 8 Fatty Acid Composition of H. pluvialis
表 8 雨生红球藻的脂肪酸组成

Biomass yield varied significantly across the different temperature and nitrate concentration conditions. At 20 °C, the cultures demonstrated the highest biomass yield, particularly at a potassium nitrate concentration of 0.21 g/L, reaching 0.665 ± 0.200 g/L. This condition aligns with optimal growth parameters reported in previous studies [13]. In contrast, biomass yields decreased under both suboptimal (10 °C) and stress-induced (30 °C) temperatures, with reductions more pronounced at higher nitrate concentrations. For instance, at 10 °C, the maximum biomass yield was 0.410 ± 0.160 g/L, while at 30 °C, it declined further to 0.350 ± 0.140 g/L.
生物质产量在不同的温度和硝酸盐浓度条件下显著变化。在 20°C 时，培养物表现出最高的生物质产量，特别是在硝酸钾浓度为 0.21 g/L 时，达到 0.665 ± 0.200 g/L。这一条件与之前研究中报道的最佳生长参数一致[13]。相比之下，在次优（10°C）和应激（30°C）温度下，生物质产量有所下降，且在较高硝酸盐浓度下下降更为显著。例如，在 10°C 时，最大生物质产量为 0.410 ± 0.160 g/L，而在 30°C 时，进一步下降至 0.350 ± 0.140 g/L。

Lipid productivity displayed an inverse relationship with nitrate concentration, consistent with the known metabolic response of H. pluvialis under nitrogen stress. At 20 °C, the highest lipid content was observed in nitrate-free conditions, reaching 46.31 ± 0.026% of dry biomass. This trend was evident across all temperatures, although the magnitude of lipid accumulation was significantly higher at 20 °C compared to 10 °C and 30 °C. At 10 °C, lipid productivity peaked at 30.12 ± 0.020%, while at 30 °C, it reached 35.25 ± 0.018%.
脂质产量与硝酸盐浓度呈反比关系，这与**雨生红球藻**（H. pluvialis）在氮胁迫下的已知代谢反应一致。在 20°C 条件下，无硝酸盐环境中观察到最高的脂质含量，达到干生物量的 46.31 ± 0.026%。这一趋势在所有温度下都很明显，但脂质积累的幅度在 20°C 时显著高于 10°C 和 30°C。在 10°C 时，脂质产量最高为 30.12 ± 0.020%，而在 30°C 时达到 35.25 ± 0.018%。

A synthesis of the findings reveals distinct trends in how temperature and nitrate concentration jointly influenced biomass and lipid productivity:
综合研究结果表明，温度和硝酸盐浓度共同影响生物量和脂质产量的规律具有显著差异：

Temperature effects  温度影响

At 20 °C, cultures consistently exhibited a balance between biomass yield and lipid productivity, making it the most favorable condition for dual optimization.
在 20°C 时，培养物表现出生物质产量和脂质生产率之间的平衡，使其成为双重优化的最佳条件。

Suboptimal temperatures (10 °C) limited metabolic activity, resulting in reduced biomass yield and lipid productivity.
在次优温度（10°C）下，代谢活动受到限制，导致生物质产量和脂质生产率降低。

Stress-induced conditions (30 °C) triggered lipid accumulation but significantly impaired biomass growth due to thermal stress on cellular enzymes [18].
应激性条件（30°C）会诱导脂质积累，但由于热应激对细胞酶的影响，显著抑制了生物质的生长[18]。

Nitrate concentration effects
硝酸盐浓度影响

High nitrate concentrations supported biomass growth but suppressed lipid synthesis, indicating that nitrogen availability redirects metabolic flux toward cell division rather than lipid storage [11].
高硝酸盐浓度促进了生物量的增长，但抑制了脂质的合成，这表明氮的供应将代谢通量引导至细胞分裂，而非脂质储存 [11]。

Nitrogen starvation (0 g/L) induced a metabolic shift favoring lipid accumulation at the expense of biomass growth. This response is consistent with previous findings where lipid biosynthesis is upregulated under nutrient-deprived conditions to serve as an energy reserve [1].
氮饥饿（0 g/L）诱导了代谢转变，表现为脂质积累增加，而生物质的增长受到抑制。这种反应与之前的研究一致，即在营养缺乏条件下，脂质的生物合成会被上调，用作能量储备 [1]。

The results demonstrate that achieving a balance between biomass and lipid productivity depends on careful calibration of temperature and nitrate levels:
研究结果表明，实现生物质与脂质生产率之间的平衡取决于对温度和硝酸盐水平的精确调控：

Optimal condition At 20 °C with 0.10 g/L nitrate, cultures achieved a synergistic balance with biomass yield of 0.560 ± 0.136 g/L and lipid productivity of 40.30 ± 0.012%.
最佳条件 在 20°C、硝酸盐浓度为 0.10 g/L 时，培养物实现了协同平衡，生物质产量为 0.560 ± 0.136 g/L，脂质生产率为 40.30 ± 0.012%。

Trade-Offs While nitrate starvation maximized lipid content, the ass°Ciated reduction in biomass yield presents a trade-off for industrial scalability. Thus, moderately low nitrate concentrations may be more suitable for balancing dual outputs.
权衡 虽然硝酸盐匮乏最大化了脂质含量，但伴随的生物质产量减少对工业规模化生产来说是一种权衡。因此，适中的低硝酸盐浓度可能更适合平衡双重产出。

Comparative analysis These findings are consistent with studies by Pereira & Otero [21] and Akter et al., [1], which reported similar trends under controlled environmental conditions. However, the superior lipid productivity at 20 °C highlights the potential for strain-specific optimization.
比较分析 这些研究结果与 Pereira & Otero [21] 和 Akter 等人 [1] 在受控环境条件下报告的相似趋势一致。然而，在 20°C 下的更高脂质生产率突出了菌株特异性优化的潜力。

The observed increase in lipid production under nitrogen starvation aligns with the metabolic strategy of microalgae to store energy under stress conditions. This is in agreement with studies by Liu et al. [18] and Fidalgo et al. [13], which highlight the role of TAG biosynthesis during nitrogen deprivation. However, the superior lipid accumulation at 20 °C suggests that the interplay of temperature and nitrate levels can be leveraged to enhance productivity beyond what is achievable under single-factor optimizations.
在氮饥饿条件下，观察到的脂质产量增加符合微藻在压力条件下储存能量的代谢策略。这与 Liu 等人[18]和 Fidalgo 等人[13]的研究一致，这些研究强调了氮缺乏期间三酰基甘油（TAG）生物合成的作用。然而，在 20°C 下的更高脂质积累表明，温度和硝酸盐水平的相互作用可以被利用，以提高生产力，超越单一因素优化所能实现的水平。

Discussion & conclusion  讨论与结论

With the addition of 1 g, H. pluvialis can be performed five times at temperature adjustments of 10 °C, 20 °C, and 30 °C.Without nitrate, g/L, 0.5 g/L, 0.25 g/L, and 0.125 g/L NaNO3. Various groups were formed and cultivated. The goal was to ascertain the variety of the culture media, the nitrate algae ranges, and the quantity of lipids.
通过添加 1 克 H. pluvialis，可以在 10°C、20°C 和 30°C 的温度条件下进行五次实验。在没有硝酸盐的情况下，使用 0 g/L、0.5 g/L、0.25 g/L 和 0.125 g/L 的 NaNO3 培养液进行分组和培养。目标是确定培养基的多样性、硝酸盐与藻类的范围以及脂质的含量。

According to research by Boussiba [3], algae are usually orange or nearly orange in the lab, even though they appear green there. According to reports, the lipid created is the cause of this hue shift, and similar marketing has been done in close developments where water is stored and lipid builds up. The fifth group of nitrate algae in this auction began to turn orange on the tenth day at 10 °C, on the eighth day at 20 °C, and on the seventh day at 30 °C. The color then spontaneously dissipated. Astahaxanthin could be the cause of the accumulation of algae coloring changes as the temperature rises. In their investigation into how production temperature affected H. pluvialis cellular development,
根据 Boussiba 的研究[3]，在实验室中，藻类通常呈现橙色或接近橙色，即使在实验室环境中它们通常为绿色。据报道，这种颜色变化是由脂质的产生引起的。在一些类似的研究中，发现储水环境中脂质的积累也导致了这种现象。在本次实验中，第五组硝酸盐培养的藻类在 10°C 条件下第 10 天开始变橙色，在 20°C 条件下第 8 天开始变橙色，在 30°C 条件下第 7 天开始变橙色。随后，这种颜色会自然消退。随着温度的升高，藻类色素变化可能与虾青素（Astaxanthin）的积累有关。在研究温度对 H. pluvialis 细胞生长的影响时，发现了这种现象。

Fábregas et al., [12],found that 25–30 °C was the ideal temperature and that 32 °C inhibited development. Roessler, [22] according to research by researchers like, 25 °C is the ideal temperature for H. pluvialis. According to [10], algae could withstand 30 °C, however their growth slowed and they went into the stationary phase sooner. Similar to other research in the literature, this study employed temperatures of 10 °C, 20 °C, and 30 °C to create an adverse environment that increased the lipid content. The study's findings showed that H. pluvialis algae grew best in units set at 20 °C, although those produced in culture media set at 30 °C reached the stationary phase sooner than those produced at 20 °C. Lipid synthesis was shown to be lower at 10 °C than at other temperatures.
Fábregas 等人[12]发现，25–30 °C 是理想温度，而 32 °C 会抑制生长。根据 Roessler[22]及其他研究人员的研究，25 °C 是雨生红球藻（H. pluvialis）的最佳生长温度。据[10]报道，藻类可以耐受 30 °C，但其生长速度减慢，并更早进入稳定期。与文献中的其他研究类似，本研究采用了 10 °C、20 °C 和 30 °C 的温度来创造不利环境，以促进脂质含量的增加。研究结果表明，雨生红球藻在 20 °C 条件下生长最佳，而在 30 °C 培养基中生长的藻类比在 20 °C 条件下更早进入稳定期。在 10 °C 条件下，脂质合成低于其他温度。

In their investigations into how lighting affects hydrocarbons, [14] and Roessler [22] found that 20,490- 40,980 Lux was the ideal light intensity for H. pluvialis. While Zhekisheva [29]; observed that the algae increased its biomass in 5 days at 10,245 Lux light intensity and in 2.5 days at 122,940 Lux light intensity, Wong et al., [27] showed that lipid production reduced under low light. In their study on light intensity, Wong et al., [27] found that lipid content dramatically dropped at illumination levels above 68,300Lux, that algal development accelerated at illumination levels above 68,300 Lux, and that algal development slowed down at illumination levels below 20,490 lx. The light intensity utilized in this investigation was 3750 μmol photons m⁻² s⁻¹ (or, approximatelly 20,490 Lux.) and it was observed that development was normal under these conditions.
在研究光照对碳氢化合物影响的过程中，[14] 和 Roessler [22] 发现，20,490-40,980 Lux 是 H. pluvialis 的理想光照强度。而 Zhekisheva [29] 观察到，在 10,245 Lux 的光照强度下，藻类在 5 天内增加了生物量；在 122,940 Lux 的光照强度下，生物量在 2.5 天内增加。Wong 等人 [27] 的研究表明，在低光条件下脂质产量减少。在对光强进行研究时，Wong 等人 [27] 发现，当光照强度超过 68,300 Lux 时，脂质含量显著下降；当光照强度超过 68,300 Lux 时，藻类生长加速；当光照强度低于 20,490 Lux 时，藻类生长减缓。本研究中使用的光强为 3750 μmol photons m⁻¹ s⁻¹（约合 20,490 Lux），结果表明在此条件下生长正常。

In their study on pH, Dayananda et al. [9] found that biomass was 0.85 g/L at pH 7.5, where the best development took place. The greatest biomass was determined to be 0.564 g/L at 20 °C in this investigation, when pH was tested between 7.0 and 7.5. This variation in biomass could result from elements like labor hours and the nutritional environment.
在关于 pH 的研究中，Dayananda 等人[9]发现，在 pH 7.5 时，生物质为 0.85 g/L，这是最佳生长条件。在本次研究中，当 pH 值在 7.0 到 7.5 之间测试时，最大生物质被确定为 0.564 g/L（在 20°C 下）。这种生物质的差异可能是由于工作时间和营养环境等因素造成的。

Reducing the amount of nitrate given to the nutritional medium has been shown in numerous experiments to increase the amount of lipids produced by algae and decrease chlorophyll a [6, 7]. The development of algae and the amount of lipids they accumulate under limited nitrogen conditions were investigated in the study “The effect of nitrogen limitation on the development and lipid composition of green algae H. pluvialis by Cerón et al. [6]. It was found that the total lipid content increased by 21%.
减少营养培养基中硝酸盐的含量已被多项实验证明可以增加藻类产生的脂质含量，同时降低叶绿素 a 的含量[6, 7]。在 Cerón 等人的研究《氮限制对绿藻 H. pluvialis 的发育和脂质组成的影响》中，研究了藻类在氮限制条件下的生长情况及其脂质积累量[6]。研究发现，总脂质含量增加了 21%。

The amount of lipids produced by the algae at each nitrate level was measured in this study after five different nitrate concentrations were given to the nutrient medium. According to the study's findings, the first group’s lipid content in the experiment at 10 °C was 10.16 ± 0.039% of dry weight, the second group's was 18.00 ± 0.010% of dry weight, the third group’s was 19.09 ± 0.010% of dry weight, the fourth group’s was 28.37 ± 0.085% of dry weight, and the fifth group's was 46.90 ± 0.015% of dry weight. First group results in the experiment at 20 °C were 15.84 ± 0.517% of dry weight, second group result was 33.31 ± 0.014% of dry weight, third group result was 24.44 ± 0.088% of dry weight, fourth group result was 45.30 ± 0.012% of dry weight, and fifth group result was 56.31 ± 0.026% of dry weight.
在这项研究中，通过向营养培养基中提供五种不同浓度的硝酸盐，测量了每种硝酸盐水平下藻类产生的脂质量。研究结果显示，在 10°C 条件下，实验第一组的脂质含量为干重的 10.16 ± 0.039%，第二组为干重的 18.00 ± 0.010%，第三组为干重的 19.09 ± 0.010%，第四组为干重的 28.37 ± 0.085%，第五组为干重的 46.90 ± 0.015%。在 20°C 条件下，第一组的脂质含量为干重的 15.84 ± 0.517%，第二组为干重的 33.31 ± 0.014%，第三组为干重的 24.44 ± 0.088%，第四组为干重的 45.30 ± 0.012%，第五组为干重的 56.31 ± 0.026%。

The first group result in the experiment at 20 °C was 15.84 ± 0.517% of dry weight, second group result was 33.31 ± 0.014% of dry weight, third group result was 24.44 ± 0.088% of dry weight, fourth group result was 45.30 ± 0.012% of dry weight, and fifth group result was 56.31 ± 0.026% of dry weight.
在 20°C 实验中，第一组的结果为 15.84 ± 0.517%干重，第二组的结果为 33.31 ± 0.014%干重，第三组的结果为 24.44 ± 0.088%干重，第四组的结果为 45.30 ± 0.012%干重，第五组的结果为 56.31 ± 0.026%干重。

First group result was 14.56 ± 0.295% of dry weight, second group result was 22.81 ± 0.014% of dry weight, third group result was 26.54 ± 0.014% of dry weight, fourth group result was 35.36 ± 0.010% of dry weight, and fifth group result aws 55.14 ± 0.015% of dry weight in the experiments at 30 °C.
在实验中，第一组的结果是干重的 14.56 ± 0.295%，第二组的结果是干重的 22.81 ± 0.014%，第三组的结果是干重的 26.54 ± 0.014%，第四组的结果是干重的 35.36 ± 0.010%，第五组的结果是干重的 55.14 ± 0.015%（实验温度为 30°C）。

According to the data, the fourth group, which was cultivated in the nutrient medium with the least amount of nitrate, and the fifth group, which had no nitrate, produced the most lipids in the trials carried out at all three temperatures. Similar to earlier research, these findings provide credence to the idea that lipid formation rises as nitrate additions to the nutritional medium fall.
根据数据，第四组（在含最少硝酸盐的培养基中培养）和第五组（在不含硝酸盐的情况下培养）在所有三个温度下的试验中产生了最多的脂质。与早期的研究类似，这些结果支持了这样的观点：随着培养基中硝酸盐的减少，脂质的生成量会增加。

On the second, sixth, twelfth, and twentieth days, samples were collected from the biomass grown in Bristol solution, and oil extractions were carried out utilizing microwaves. Fatty acids derived from the study’s biomast on day 20Hexadekadienoic acid 27.9, hexadekatrienoic acid 2.1, margaric acid 1.6, searic acid 2.0, oleic acid 46.0, linoleic acid 7.4, linolenic acid 9.1, ecocenoic acid 0, arachidic acid 0, behenic acid 0, lignoceric acid 0, mystoleic acid 0.8, mystoleic acid 0.6, pentadekanoic acid 0.5, pentadekanoic acid 0.5, and palmitic acid 27.8 and palmitoleic acid 1.8.To ascertain the lipid contents of H.pluvialismicroalgae, [6] employed nutritional media with control, 34 mM, and 85 mM salinity. The control group had the following fatty acid compositions: 1.14 percent palmitic acid, 10.64 percent palmitoleic acid, 28.19 percent stearic acid, 13.35 percent oleic acid, 22.12 percent linoleic acid, trace amounts of behenic acid, 10.54 percent erucic acid, and trace amounts of lignoceric acid.
在第 2 天、第 6 天、第 12 天和第 20 天，从在布里斯托溶液中生长的生物质中采集样本，并利用微波进行了油脂提取。在第 20 天研究的生物质中提取的脂肪酸包括：十六碳二烯酸 27.9%、十六碳三烯酸 2.1%、十七烷酸 1.6%、硬脂酸 2.0%、油酸 46.0%、亚油酸 7.4%、亚麻酸 9.1%、二十碳烯酸 0%、花生酸 0%、二十二烷酸 0%、二十四烷酸 0%、肉豆蔻烯酸 0.8%、肉豆蔻酸 0.6%、十五烷酸 0.5%（两次出现），棕榈酸 27.8%和棕榈油酸 1.8%。 为了确定 H. pluvialis 微藻的脂质含量，[6]使用了控制组、34 mM 和 85 mM 盐度的营养培养基。控制组的脂肪酸组成如下：棕榈酸 1.14%、棕榈油酸 10.64%、硬脂酸 28.19%、油酸 13.35%、亚油酸 22.12%、痕量的二十二烷酸、芥酸 10.54%以及痕量的二十四烷酸。

Trace levels of the following fatty acids were detected in the algae grown in nutritional medium with a salinity of 34 mM: palmitic acid 25.62, palmitoleic acid 14.84, stearic acid 5.91, oleic acid 25.23, linoleic acid 19.15, behenic acid 1.88, erucic acid 7.30, and lignoceric acid. Palmitic acid was 34.76, trace levels of palmitoleic acid, stearic acid 9.33, oleic acid 28.28, linoleic acid 10.06, behenic acid 2.94, trace amounts of erucic acid, and lignoceric acid 15.61 in algae grown in nutritional medium containing 85 mM NaCl. The percentages in this study were as follows: oleic acid 59.04, palmitic acid 16.62, methyl Cis 11, 14, 17, ecosatrienoic acid 0.23, myristic acid 0.31, behenic acid 0.41, ecocenoic acid 1.01, linoleic acid 9.92, linolenic acid 9.50, margaric acid 0.28, pentadekanoic acid 0.18, and stearic acid 2.50.
在盐度为 34 mM 的营养培养基中生长的藻类中检测到以下脂肪酸的微量含量：棕榈酸 25.62、棕榈油酸 14.84、硬脂酸 5.91、油酸 25.23、亚油酸 19.15、二十二酸 1.88、芥酸 7.30 和木蜡酸。 在含 85 mM NaCl 的营养培养基中生长的藻类中，棕榈酸为 34.76，棕榈油酸微量，硬脂酸 9.33，油酸 28.28，亚油酸 10.06，二十二酸 2.94，芥酸微量，木蜡酸 15.61。 本研究中的百分比如下：油酸 59.04、棕榈酸 16.62、甲基顺-11,14,17-二十碳三烯酸 0.23、肉豆蔻酸 0.31、二十二酸 0.41、二十碳烯酸 1.01、亚油酸 9.92、亚麻酸 9.50、十七酸 0.28、十五烷酸 0.18 和硬脂酸 2.50。

The fourth group, which was cultivated at 20 °C and in a nutrient medium containing 0.25 g NaNO₃, was shown to be the most effective group in terms of oil production for H.pluvialis in the studies conducted to enhance the amount of oil of algae. The most optimal groups were used to conduct intensive cultural investigations.
第四组在 20°C 下培养，并在含有 0.25 g NaNO₃ 的培养基中生长，被证明是提高雨生红球藻 (H. pluvialis) 油脂产量最有效的一组。在研究中，最优的组别被用于深入的培养实验。

Experiments at all three temperatures revealed that whereas lipid production was inversely proportional to both the increase in biomass and the increase in sodium nitrate, the increase in algal biomass was directly linked to the latter.
在所有三个温度下的实验表明，脂质生产与生物量的增加以及硝酸钠浓度的增加呈负相关，而藻类生物量的增加则与硝酸钠浓度的增加呈正相关。

On the fifth day of the 10 °C studies, the fourth day of the 20 °C investigations, and the third day of the 30 °C experiments, it was seen that the algae began to become orange. According to earlier research, as the algae began to become orange, the rates of astaxanthin and oil rose [2, 19].
在 10°C 实验的第五天、20°C 研究的第四天以及 30°C 实验的第三天，观察到藻类开始变成橙色。根据早期的研究，当藻类开始变成橙色时，虾青素和油脂的含量会增加 [2, 19]。

The fatty acid profile in H. pluvialis was similar under control and stress conditions and revealed that palmitic, estearic, oleic, linoleic, linolenic and linolelaidic acids were the major components (Damini et al., 2010; [26]).
在控制条件和胁迫条件下，小球藻（H. pluvialis）的脂肪酸组成相似，研究显示棕榈酸、硬脂酸、油酸、亚油酸、亚麻酸和反式亚油酸是主要成分（Damini 等, 2010; [26]）。

After 3 days of light-induced stress, the content of both bioactive lipids significantly increased compared to controls. Palmitic, linoleic, and α linolenic fatty acid content was higher whereas caproic acid content diminished in H. pluvialis under stress [6]. The limitations of this study are as follows:
在光诱导胁迫 3 天后，与对照组相比，两种生物活性脂质的含量均显著提高。在胁迫条件下，小球藻中的棕榈酸、亚油酸和α-亚麻酸含量增加，而己酸含量减少 [6]。本研究的局限性如下：

Scale of experimentation The study was conducted in laboratory-scale cultivation systems (e.g., Erlenmeyer flasks), which may not fully replicate the complexities of industrial-scale operations. Factors like aeration dynamics, light penetration, and nutrient distribution in larger systems can significantly impact results.
实验规模 该研究是在实验室规模的培养系统（例如锥形瓶）中进行的，这可能无法完全反映工业规模操作的复杂性。诸如通气动态、光穿透和营养分布等因素在更大规模的系统中可能会对结果产生显著影响。

Limited environmental variables The study focused on temperature and nitrate concentration as the primary variables. While these are critical factors, other influential variables, such as light intensity, salinity, and pH, were held constant and not explored in detail, which could limit the comprehensiveness of the findings.
环境变量有限 本研究将温度和硝酸盐浓度作为主要变量进行研究。尽管这些是关键因素，但其他具有影响力的变量（如光照强度、盐度和 pH 值）被保持不变，未作详细探讨，这可能会限制研究结果的全面性。

Static cultivation conditions The experiments were conducted under static conditions, with fixed nitrate levels and constant temperatures throughout the cultivation period. Dynamic conditions, such as fluctuating temperatures or staged nutrient depletion, which are common in real-world industrial settings, were not evaluated.
静态培养条件 实验在静态条件下进行，在整个培养过程中硝酸盐水平固定，温度恒定。未评估真实工业环境中常见的动态条件，例如温度波动或分阶段的养分消耗。

Short cultivation duration The cultivation period was limited to 10 days, which may not fully capture the long-term effects of the tested variables on biomass yield and lipid productivity. Industrial cultivation often spans longer durations, which can introduce additional challenges like contamination and nutrient exhaustion.
培养周期较短 培养周期仅为 10 天，这可能无法充分反映所测试变量对生物质产量和脂质生产力的长期影响。工业化培养通常持续更长时间，这可能会带来额外的挑战，例如污染和养分耗尽。

Lack of economic analysis While the study identifies optimal conditions for dual productivity, it does not include a techno-economic analysis or cost assessment to evaluate the financial feasibility of implementing these conditions at scale.
缺乏经济分析 尽管该研究确定了双重产能的最佳条件，但并未包含技术经济分析或成本评估，以评估在大规模实施这些条件的财务可行性。

Single strain usage The study focuseson a specific strain of Haematococcus pluvialis. The findings may not be directly applicable to other strains or species of microalgae, which could exhibit different responses to the same environmental conditions.
**单一菌株的使用** 本研究集中于一种特定菌株的雨生红球藻。研究结果可能无法直接适用于其他菌株或微藻物种，因为它们在相同的环境条件下可能表现出不同的反应。

Absence of stress-inducing factors beyond temperature and nitrate Other stressors that can enhance lipid or astaxanthin accumulation, such as high light intensity or oxidative stress, were not explored. Incorporating such factors could further optimize productivity.
**除温度和硝酸盐外缺乏应激诱导因素** 本研究未探讨其他可能促进脂质或虾青素积累的应激因素，例如高光强或氧化应激。引入这些因素可能进一步优化生产力。

Harvesting and downstream processing The study primarily emphasizes cultivation conditions and does not address challenges associated with harvesting biomass or lipid extraction at industrial scales, such as energy consumption and process efficiency.
### 收获与下游加工 该研究主要关注培养条件，但未讨论与工业规模下收获生物质或提取脂质相关的挑战，例如能源消耗和工艺效率。

Limited real-world validation The study’s results have not been validated in pilot-scale or field settings, where variables such as weather, contamination, and resource limitations can significantly impact outcomes.
### 现实验证的局限性 该研究的结果尚未在中试规模或实际环境中验证。在这些情况下，天气、污染和资源限制等变量可能会对结果产生显著影响。

Environmental impact considerations While the study identifies sustainable cultivation practices, it does not evaluate the environmental footprint of these practices, such as water usage, energy demand, or carbon emissions.
### 环境影响的考量 尽管研究确定了可持续的培养方法，但并未评估这些方法的环境足迹，例如水资源使用、能源需求或碳排放等方面的影响。

Addressing these limitations in future research, such as expanding the range of environmental variables, testing dynamic cultivation conditions, and conducting pilot-scale validations, could enhance the applicability and scalability of the findings. Additionally, incorporating economic and environmental impact assessments would provide a more comprehensive understanding of the feasibility of implementing these cultivation strategies on an industrial scale.
在未来的研究中解决这些局限性，例如扩大环境变量的范围、测试动态培养条件以及进行中试规模的验证，可能会提升研究结果的适用性和可扩展性。此外，结合经济和环境影响评估将有助于更全面地了解在工业规模上实施这些培养策略的可行性。

Economic feasibility analysis for scaling Haematococcus pluvialis cultivation involves evaluating costs, revenues, and profitability to determine the viability of industrial implementation. Costs can be divided into capital expenditures (CAPEX) and operational expenditures (OPEX). CAPEX includes infrastructure setup, equipment purchase for photobioreactors or open pond systems, land acquisition, and construction costs. OPEX encompasses recurring expenses such as nutrients, energy for lighting and temperature regulation, labor, water usage, and maintenance. Harvesting and lipid extraction processes also contribute significantly to operational costs.
规模化栽培雨生红球藻（*Haematococcus pluvialis*）的经济可行性分析涉及评估成本、收入和盈利能力，以确定工业化实施的可行性。成本可分为资本支出（CAPEX）和运营支出（OPEX）。CAPEX 包括基础设施建设、光生物反应器或开放池系统的设备采购、土地获取以及施工成本。OPEX 包括营养物质、用于照明和温度调节的能源、人工、水资源使用和维护等经常性开支。收获和脂质提取过程也对运营成本有显著贡献。

Revenue streams primarily consist of earnings from lipid production as a biofuel feedstock. Co-products such as astaxanthin, proteins, and pigments can enhance profitability by diversifying the product portfolio. Additional revenue may be generated through carbon credits if CO₂ sequestration technologies are integrated and waste biomass is valorized as animal feed or fertilizers. Comparing these costs and revenues using methods such as net present value (NPV), internal rate of return (IRR), and payback period calculations provides a clear picture of economic feasibility. NPV calculates the present value of net earnings over the project’s lifetime, IRR identifies the profitability rate, and the payback period estimates the time required to recover initial investments.
收入来源主要包括将脂质作为生物燃料原料的收益。副产品如虾青素、蛋白质和色素等可以通过丰富产品组合来提高盈利能力。如果整合了二氧化碳（CO₂）封存技术，并将废弃生物质转化为动物饲料或肥料，还可以产生额外收入。通过使用净现值（NPV）、内部收益率（IRR）和回收期等方法比较这些成本和收益，可以清晰地评估经济可行性。NPV 计算项目生命周期内净收益的现值，IRR 确定项目的盈利率，而回收期估算收回初始投资所需的时间。

Scalability must also be considered, as larger operations benefit from economies of scale, reducing per-unit costs for equipment, materials, and labor. Process efficiency improvements, such as optimizing lipid yields, further enhance economic viability. However, the scalability of H. pluvialis cultivation is influenced by external factors such as market demand for algal lipids and co-products, nutrient availability, and energy costs. Sensitivity analysis should be performed to evaluate how fluctuations in these parameters impact profitability, such as variations in potassium nitrate prices, electricity rates, or lipid market prices. Additionally, changes in lipid yield under real-world conditions must be factored into the analysis.
还必须考虑可扩展性，因为规模更大的运营能够通过规模经济效益降低设备、材料和人工的单位成本。通过优化脂质产量等提升工艺效率，也能进一步提高经济可行性。然而，**雨生红球藻**（H. pluvialis）培育的可扩展性还受到外部因素的影响，如藻类脂质及副产品的市场需求、养分供应的可用性和能源成本等。应进行敏感性分析，评估这些参数波动对盈利能力的影响，例如硝酸钾价格、电费或脂质市场价格的变化。此外，还需将脂质产量在真实环境条件下的变化纳入分析。

A life cycle assessment (LCA) can complement economic analysis by incorporating environmental and sustainability metrics, such as water and energy footprints, waste management practices, and carbon emissions. Combining economic feasibility with LCA ensures that scaling operations are both financially and environmentally sustainable. Practical implementation should begin with pilot-scale validations to refine cost estimates and optimize processes. Partnerships with biofuel companies or government subsidies can offset initial costs and enhance financial viability. Diversifying revenue streams by targeting co-product markets reduces dependency on biofuel prices alone, ensuring long-term profitability. By addressing these factors, the economic feasibility of H. pluvialis cultivation for industrial applications can be robustly assessed and optimized.
生命周期评估（LCA）可以补充经济分析，将环境和可持续性指标纳入考量，如水和能源足迹、废物管理实践以及碳排放等。将经济可行性与 LCA 相结合，可以确保扩大生产既符合财务需求，也符合环境可持续性要求。实际实施应从试验规模的验证开始，以优化工艺并精确估算成本。与生物燃料公司合作或利用政府补贴可以抵消初始成本，提高财务可行性。通过开拓副产品市场来实现收入来源多样化，可以减少对生物燃料价格的依赖，从而确保长期盈利能力。通过解决上述因素，可以全面评估和优化雨生红球藻在工业应用中的经济可行性。

This study systematically investigates the interactive effects of temperature and nitrate concentration on Haematococcus pluvialis, contributing significantly to the existing body of research on microalgal biomass and lipid productivity. In the broader context of microalgal research, optimizing environmental parameters has been a recurring theme due to its critical role in enhancing productivity and scalability. The findings align with and expand upon several key studies in the literature, offering new insights into strain-specific responses and industrial applications.
本研究系统性地探讨了温度和硝酸盐浓度对雨生红球藻的交互作用，为微藻生物质和脂质生产力的现有研究做出了重要贡献。在微藻研究的更广泛背景下，优化环境参数一直是一个反复出现的主题，因为它在提高生产力和可扩展性方面起着关键作用。研究结果与文献中的若干关键研究一致，并进一步拓展了这些研究，提供了关于菌株特异性反应和工业应用的新见解。

Nitrogen availability is widely recognized as a primary driver of lipid accumulation in microalgae. Fidalgo et al. [13] demonstrated that nitrogen starvation redirects metabolic flux from cellular proliferation to triacylglycerol (TAG) synthesis, resulting in enhanced lipid accumulation in H. pluvialis. This mechanism was similarly observed in this study, where nitrogen-deprived conditions (0 g/L nitrate) led to the highest lipid productivity. However, Fidalgo et al. [13] focused primarily on nitrogen as an isolated variable, while the present study introduces temperature as an additional factor, highlighting its synergistic effects. Specifically, lipid content was maximized at 20 °C, emphasizing the importance of maintaining optimal thermal conditions alongside nutrient manipulation.
氮的可用性被广泛认为是驱动微藻脂质积累的主要因素。Fidalgo 等人 [13] 证明，氮饥饿会将代谢流从细胞增殖转向三酰基甘油（TAG）合成，从而导致 H. pluvialis 中脂质积累的增加。这一机制在本研究中也得到了类似的观察，在缺氮条件（0 g/L 硝酸盐）下，脂质生产率达到最高。然而，Fidalgo 等人 [13] 主要将氮作为单一变量进行研究，而本研究则引入了温度作为额外因素，突出了其协同效应。具体而言，在 20 °C 时脂质含量达到最大化，这强调了在营养调控的同时维持最佳温度条件的重要性。

Akter et al., [1] explored nitrate depletion and its influence on dual productivity (biomass and lipid), observing that nitrate-starved conditions enhance lipid accumulation but at the expense of biomass yield. The current study corroborates this finding and further identifies conditions, such as 0.10 g/L nitrate at 20 °C, where a balance between biomass yield (0.560 ± 0.136 g/L) and lipid content (40.30 ± 0.012% dry weight) can be achieved. This dual optimization provides valuable insights for industrial scalability, bridging the gap between purely laboratory focused studies and practical applications.
Akter 等人 [1] 探讨了硝酸盐消耗及其对双重产量（生物质和脂质）的影响，发现缺乏硝酸盐的条件会促进脂质积累，但以牺牲生物质产量为代价。本研究证实了这一发现，并进一步确定了在例如 20°C 下硝酸盐浓度为 0.10 g/L 的条件下，可实现生物质产量（0.560 ± 0.136 g/L）和脂质含量（40.30 ± 0.012% 干重）之间的平衡。这种双重优化为工业规模化提供了宝贵的见解，弥合了纯实验室研究与实际应用之间的差距。

Temperature, as an environmental factor, has been extensively studied for its impact on enzymatic activity and metabolic pathways in microalgae. Pereira & Otero [21] observed that H. pluvialis achieves peak biomass productivity at moderate temperatures around 20 °C, with significant reductions in growth observed at both low (10 °C) and high (30 °C) temperatures due to suboptimal enzyme functionality and thermal stress. These findings align closely with the present study, which identifies 20 °C as the most favorable temperature for dual productivity optimization. Furthermore, the current research highlights that while high temperatures (30 °C) induce lipid accumulation, they significantly compromise biomass yield, underscoring the importance of balancing these factors in industrial systems.
温度作为一种环境因素，对于酶活性和微藻代谢途径的影响已被广泛研究。Pereira 和 Otero [21] 观察到，H. pluvialis 在约 20°C 的中等温度下实现了最高的生物量生产率，而在低温（10°C）和高温（30°C）下，由于酶功能不佳和热应激，生长显著下降。这些发现与本研究高度一致，本研究确定 20°C 是双重生产率优化的最佳温度。此外，当前研究还指出，高温（30°C）虽然会诱导脂质积累，但会显著降低生物量产量，这凸显了在工业系统中平衡这些因素的重要性。

The influence of temperature and nitrate on microalgae has also been explored in species beyond H. pluvialis. For instance, Guschina and Harwood [15] reported that in Chlorella vulgaris, nitrogen starvation leads to lipid accumulation, but the metabolic pathways differ significantly from those in H. pluvialis, reflecting species-specific responses. This underscores the need for targeted optimization strategies, as demonstrated in this study, to maximize the potential of individual microalgal strains. Similarly, [27], emphasized the role of light intensity and carbon availability in influencing microalgal productivity. While the present study held these variables constant, integrating them into future investigations could provide a more comprehensive understanding of environmental interactions.
温度和硝酸盐对微藻的影响也在 H. pluvialis 之外的其他物种中得到了研究。例如，Guschina 和 Harwood [15] 报道，在小球藻（*Chlorella vulgaris*）中，氮饥饿会导致脂质积累，但与 H. pluvialis 相比，其代谢途径存在显著差异，这反映了物种特异性的响应。这进一步强调了针对性优化策略的必要性，正如本研究所展示的，以最大化单个微藻菌株的潜力。同样，[27] 强调了光强和碳源可用性在影响微藻生产力中的作用。虽然本研究将这些变量保持恒定，但在未来的研究中将其纳入考量可能会更全面地揭示环境交互关系。

Liu et al. [18] introduced the concept of dual optimization, focusing on achieving high lipid productivity without sacrificing biomass yield. While their work primarily addressed dynamic environmental conditions, the current study provides a systematic evaluation of static cultivation parameters, offering complementary insights. The identification of optimal conditions in this study provides a foundation for further exploration into dynamic or staged cultivation strategies, where nitrate levels and temperatures are adjusted throughout the growth cycle to maximize productivity.
Liu 等人[18] 引入了双重优化的概念，重点是在不牺牲生物质产量的情况下实现高脂质生产力。虽然他们的研究主要针对动态环境条件，本研究则系统性地评估了静态培养参数，提供了互补性的见解。本研究中最佳条件的确定为进一步探索动态或分阶段培养策略奠定了基础，在整个生长周期中调节硝酸盐水平和温度以最大化生产力。

From an industrial perspective, the scalability of H. pluvialis cultivation remains a critical challenge. Caltzontzin-Rabell et al. [5] emphasized the importance of integrating findings from laboratory studies into pilot-scale systems, highlighting barriers such as contamination, resource allocation, and cost-efficiency. The present study addresses this gap by focusing on conditions that balance productivity metrics, making them more suitable for large-scale applications. Additionally, the study’s emphasis on moderate nitrate depletion and optimal temperatures aligns with Mota et al. [20], who suggested that achieving such balances is crucial for cost-effective production of high-value products like astaxanthin and biofuels.
从工业角度来看，雨生红球藻（H. pluvialis）培养的可扩展性仍然是一个关键挑战。Caltzontzin-Rabell 等人[5] 强调了将实验室研究成果整合到中试规模系统中的重要性，并指出了污染、资源分配和成本效益等障碍。本研究通过关注在生产力指标之间取得平衡的条件来填补这一空白，使其更适合大规模应用。此外，研究对适度硝酸盐消耗和最佳温度的强调，与 Mota 等人[20] 的观点一致，他们认为实现这种平衡对于高价值产品（如虾青素和生物燃料）的成本效益生产至关重要。

While nitrogen starvation and temperature optimization are central to both this study and the broader literature, the unique contribution of this research lies in its systematic analysis of their interaction, which has not been extensively explored in prior works. By identifying conditions that enhance lipid accumulation without severely compromising biomass yield, this study provides a robust framework for the industrial cultivation of H. pluvialis. Moreover, its findings pave the way for integrating additional environmental variables, such as light and salinity, into future research.
虽然氮饥饿和温度优化是本研究以及更广泛文献的核心主题，但本研究的独特贡献在于系统分析了这两者的相互作用，而这一点在以往的研究中尚未被广泛探讨。通过确定既能增强脂质积累又不会严重影响生物量产量的条件，本研究为工业化培养**雨生红球藻**（*Haematococcus pluvialis*）提供了一个坚实的框架。此外，其研究结果为将光照、盐度等其他环境变量纳入未来研究铺平了道路。

Haematococcus pluvialis, Nannochloropsis sp., and Phaeodactylum tricornutum represent valuable microalgal species with unique lipid and fatty acid profiles, making them significant candidates for biotechnological applications. Scodelaro Bilbao et al. [23] highlight Haematococcus pluvialis as a rich source of fatty acids and phytosterols, with notable nutritional and biological implications for human health and industrial use. Similarly, Yu et al. [28] demonstrate that Nannochloropsis sp. QII produces high levels of lipids, particularly triacylglycerols, under nitrogen-limited conditions, emphasizing its potential as a sustainable biofuel feedstock. Complementing these findings, Siron et al. [25] investigate the lipid composition of Phaeodactylum tricornutum and Dunaliella tertiolecta, revealing how phosphorus deficiency and growth phases significantly influence their fatty acid profiles. Together, these studies underscore the importance of optimizing growth conditions and nutrient regimes to enhance the lipid productivity of microalgae for diverse applications in nutrition, biofuel, and aquaculture industries.
**雨生红球藻**（*Haematococcus pluvialis*）、**小球藻属**（*Nannochloropsis* sp.）和**三角褐指藻**（*Phaeodactylum tricornutum*）是具有独特脂质和脂肪酸特性的宝贵微藻种类，使其成为生物技术应用中的重要候选对象。Scodelaro Bilbao 等人 [23] 强调，**雨生红球藻**是脂肪酸和植物甾醇的丰富来源，具有显著的营养和生物学意义，对人类健康和工业应用至关重要。同样，Yu 等人 [28] 研究表明，**小球藻**（*Nannochloropsis* sp. QII）在氮限制条件下会产生高水平的脂质，尤其是三酰甘油（triacylglycerols），突显了其作为可持续生物燃料原料的潜力。补充上述发现，Siron 等人 [25] 则研究了**三角褐指藻**（*Phaeodactylum tricornutum*）和**短小叉角藻**（*Dunaliella tertiolecta*）的脂质组成，揭示了磷缺乏和生长阶段如何显著影响其脂肪酸特性。这些研究共同强调了优化生长条件和营养策略对于提高微藻脂质生产力的重要性，以满足营养、生物燃料和水产养殖行业的多元化需求。

In conclusion, the findings of this study are consistent with existing research on the effects of temperature and nitrate on microalgae, while providing new insights into their combined influence on H. pluvialis. The dual optimization strategy identified here complements the work of Fidalgo et al. [13] and Akter et al. [1] by addressing the trade-offs between lipid and biomass productivity. It also expands on the studies by Pereira & Otero [21] and Liu et al. [18] by emphasizing the scalability of optimized conditions. By bridging laboratory findings with practical applications, this research contributes significantly to the sustainable and scalable production of microalgal biofuels and high-value bioproducts.
总之，本研究的发现与现有关于温度和硝酸盐对微藻影响的研究一致，同时提供了它们对雨生红球藻(H. pluvialis)联合影响的新见解。本文提出的双重优化策略，在解决脂质与生物量生产力之间的权衡问题上，补充了 Fidalgo 等人[13]和 Akter 等人[1]的研究成果。同时，本研究在 Pereira & Otero[21]和 Liu 等人[18]的基础上进一步强调了优化条件的可扩展性。通过将实验室研究与实际应用相结合，本研究为微藻生物燃料及高价值生物产品的可持续、可扩展生产做出了重要贡献。

Future work should focus on validating the findings under pilot-scale and real-world conditions to assess scalability and practicality. Dynamic cultivation strategies, such as staged nutrient depletion and temperature adjustments, should be explored to further optimize lipid and biomass productivity over longer growth cycles. Integrating additional environmental factors, such as light intensity and CO₂ levels, could provide a more comprehensive understanding of their combined effects on Haematococcus pluvialis. Moreover, techno-economic and life cycle assessments will be essential to evaluate the cost-effectiveness and sustainability of implementing these optimized conditions at industrial scales. Expanding the study to include co-product optimization, such as astaxanthin, will also enhance the economic viability of large-scale cultivation systems.
未来的工作应重点验证这些发现是否在中试规模和实际条件下成立，以评估其可扩展性和实用性。应探索动态培养策略，例如分阶段的营养物质消耗和温度调整，以进一步优化较长生长周期内的脂质和生物质生产力。结合其他环境因素，如光照强度和二氧化碳水平，可能有助于更全面地理解其对雨生红球藻的综合影响。此外，技术经济评估和生命周期评估对于评估在工业规模下实施这些优化条件的成本效益和可持续性至关重要。扩展研究范围，包括副产品优化（如虾青素），也将提升大规模培养系统的经济可行性。