The mixed effects of temperature (20 °C, 25 °C and 30 °C), nitrate concentration (0.5 mM and 2.0 mM), pH buffer, and bicarbonate addition (trigger) on biomass growth and lipid accumulation were investigated in the environmental alga PW95 during batch experiments in standardized growth medium. PW95 was isolated from coal-bed methane production water and classified as a Chlamydomonas-like species by morphological characterization and phylogenetic analysis (18S, ITS, rbcL). A factorial experimental design tested the mixed effects on PW95 before and after nitrate depletion to determine a low cost, high efficiency combination of treatments for biomass growth and lipid accumulation. Results showed buffer addition affected growth for most of the treatments and bicarbonate trigger had no statistically significant effect on growth and lipid accumulation. PW95 displayed the highest growth rate and chlorophyll content at 30 °C and 2.0 mM nitrate and there was an inverse relation between biomass accumulation and lipid accumulation at the extremes of nitrate concentration and temperature. The combination of higher temperature (30 °C) and lower nitrate level (0.5 mM) without the use of a buffer or bicarbonate addition resulted in maximal daily biomass accumulation (5.30 × 10⁶ cells/mL), high biofuel potential before and after nitrate depletion (27% and 20%), higher biofuel productivity (16 and 15 mg/L/d, respectively), and desirable fatty acid profiles (saturated and unsaturated C16 and C18 chains). Our results indicate an important interaction between low nitrate levels, temperature, and elevated pH for trade-offs between biomass and lipid production in PW95. This work serves as a model to approach and advance the study of physiological responses of novel microalgae to diverse culture conditions that mimic environmental changes for outdoor biofuel production. The most promising conditions for growth and biofuel production were identified for PW95 and this approach can be implemented for other microalgal production systems.

在环境藻类中研究了温度（20°C，25°C和30°C），硝酸盐浓度（0.5 mM和2.0 mM），pH缓冲液和碳酸氢盐添加（触发）对生物量生长和脂质积累的混合影响。在标准生长培养基中进行批次实验时，PW95。从煤层气生产水中分离出PW95，并通过形态特征和系统发育分析将其归类为衣藻类（18S，ITS，rbcL）。析因实验设计测试了硝酸盐消耗之前和之后对PW95的混合影响，以确定用于生物质生长和脂质积累的低成本，高效处理组合。结果表明，大多数处理中添加缓冲液都会影响生长，碳酸氢盐引发剂对生长和脂质蓄积没有统计学显着影响。PW95在30°C和2.0 mM硝酸盐下表现出最高的生长速率和叶绿素含量，并且在硝酸盐浓度和温度的极端条件下，生物量积累与脂质积累呈反比关系。在不使用缓冲液或碳酸氢盐的情况下，较高的温度（30°C）和较低的硝酸盐水平（0.5 mM）的组合导致最大的每日生物量积累（5.30×10 ⁶ 细胞/ mL），硝酸盐消耗之前和之后的高生物燃料潜力（27％和20％），更高的生物燃料生产率（分别为16和15 mg / L / d）和理想的脂肪酸谱（饱和和不饱和C16和C18链） ）。我们的结果表明低硝酸盐水平，温度和pH升高之间的重要相互作用，是PW95中生物质和脂质生产之间的权衡。这项工作作为一种模式，可以接近并推动新型微藻对模仿室外生物燃料生产环境变化的多种培养条件的生理反应的研究。确定了PW95增长和生物燃料生产的最有希望的条件，并且该方法可用于其他微藻生产系统。