一种将 Euglena gracilis 适应木薯基质以实现异养栽培的方法
2026年6月12日
本方案描述了逐步将Euglena gracilis适应木薯基底物的方法，以及随后在异养栽培条件下多种木薯产品中生长、生存能力和生物量产量的评估。
我们的研究重点是将新生的Euglena gracilis叶片适应木薯废弃物，研究生长、氰化物减少和富含蛋白质的生物质生产。该方案可应用于可持续木薯加工、农业废弃物加价、饲料生产及低成本生物技术系统。首先，将无菌500毫升通风盖瓶标注为garri。
在烧瓶上标记合适的目标浓度。将复制品鉴定和计划灭菌状态标记为高压灭菌或非高压灭菌。在分析天平上放一艘称重船。
按压后，称重加里以匹配最终工作体积。将称重后的garri无菌倒入一个装有300毫升去离子或蒸馏水的500毫升无菌烧壶中。重新给每个烧瓶盖上通风盖。
将每个悬浮液混合三分钟，直到混合物均匀。如果使用磁搅拌，将烧瓶放在搅拌板上，将速度设为200克，形成浅漩涡而不溅水。让悬浮液在25摄氏度下保持不扰的状态10分钟。
发酵前立即拧紧通风盖，手动旋转烧瓶五圈，轻柔均匀地均匀混合物并重新悬浮沉积物。为准备额外的木薯制品介质，需标记瓶子并称重底材，如前所示，根据工作体积达到每升30克。将称重的底材无菌倒入装有300毫升去离子或蒸馏水的无菌瓶中。
重新给每个烧瓶盖上通风盖。像之前演示的那样，将悬浮液加水并混合。确认瓶子是否装有无菌的透气通风盖，以允许气体交换同时减少环境污染。
将烧瓶直立放置，温度为25摄氏度，放置在远离阳光直射和高流量空气的指定区域，并至少在无扰动的情况下孵育72小时。记录每个烧瓶的发酵开始日期和时间及状态。确认每个500毫升的瓶子发酵后液体不超过300毫升，以防止灭菌时沸腾。
一旦通气盖松到手指紧紧的位置，再在烧瓶上贴上高压灭菌指示带。将烧瓶放入高压灭菌器安全托盘后，将托盘放入高压灭菌器，选择P13液循环。将消毒参数设为121摄氏度，持续30分钟进行液体灭菌。
开始高压灭菌器循环。循环结束后，让高压灭菌室压力恢复到大气压，然后缓慢打开高压灭菌室门。戴上耐热手套，取出烧瓶，让它们冷却至室温。
在接种前，将冷却后的烧瓶在室温下保存一夜。直接使用发酵木薯培养基，无需高压灭菌，以保存本地微生物群落。在72小时发酵后立即接种非高压灭菌培养基，以减少环境的意外污染。
通过使用相同类型的500毫升通风盖瓶、相同的工作量和相同的发酵时长，确保所有基底条件下的处理一致性。将Euglena gracilis接种到新鲜的异养养分培养基中。在完全黑暗、28摄氏度下孵育，并在120G下持续轨道震动，直到培养物达到对数生长中段。
使用复合光学显微镜在明场模式下，以100倍至400倍总放大率观察培养物，确认其活跃运动、细胞形态延长及抗热度低。排除表现出过度细胞残骸、异常形态或适应实验前强烈要求的培养物。在通过收紧通风盖轻柔均匀培养后，用手旋转瓶子15秒，以确保细胞分布均匀且不会造成过大压力。
使用诺伊鲍尔血细胞计测定细胞密度。培养液混合完成后，将培养液旋转15秒，将一毫升培养液移入无菌的1.5毫升微离心管中。用70%乙醇清洗血细胞仪和盖子，让血细胞仪和盖子自然干燥。
将10微升培养液移入血细胞计的两个网格上，并将盖套套覆盖在湿样本上。使用10倍物镜的明场显微镜，聚焦于网格，并根据标准边界规则计数大网格中定义的格子。在第二个腔室重复细胞计数，计算每毫升的平均细胞数和细胞密度。
调整接种体积，使所有实验培养基的最终接种量体积均为5%。为每种处理条件准备独立接种剂，以保持实验组间初始细胞密度的一致性。将已处理好的E. gracilis接种剂加入高压和非高压灭菌木薯培养基中，最终加里浓度为每升10、20和30克。
在所有处理条件下使用相等的接种量，以标准化初始细胞密度。拧紧通气盖后，轻轻以圆周旋转瓶子15秒，以分布细胞。在完全黑暗、25摄氏度下孵育培养，以启动异养生长。
减少非高压灭菌培养基的搅拌，以保护本地微生物群落结构和底物层积。每天混合一次所有培养物，轻柔手动旋转15秒，以保持E. gracilis细胞与悬浮木薯物质的接触。使用无菌移液器尖端，每48小时从每个培养物中无菌采集一毫升样本。
在采样前，拧紧通风盖并轻轻旋转培养瓶15秒，以确保细胞和悬浮物均匀分布。将一小份培养物转移到无菌的1.5毫升微离心管后，根据需要用去离子水稀释样本，以改善单个细胞的可视化。通过将10微升稀释样品移液到干净的显微镜载玻片上，并用覆盖套覆盖样品，准备湿装显微镜载玻片。
使用复合光学显微镜在明场模式下，以100倍至400倍总放大率观察样本，评估细胞运动、形态及坚持频率，作为适应状态的指标。每七天，将每种培养物的体积20%无菌转移到新鲜制备的木薯培养基中，采用相同的加里浓度和灭菌条件。保持通道间的转移量一致，并重复连续传代，直到培养物在30克每升木薯培养基中表现出稳定生长、持续的运动性和低坚持频率。
在高浓度木薯适应和比较生长分析后，使用比色试纸评估氰化物水平并观察已形成的颜色。离心后，收集生物质进行下游分析。在完全暗耕条件下，以每升30克加里种植的培养物表现出比低浓度更高的综合生长。
加里浓度较低（每升10至20克）显示可检测但生长较小且变化更大。40至50克/升的高浓度细胞密度达到峰值，但稳定延迟且变异性更大。锥蓝排除检测显示，维持在每升30克加里的培养物在抽样时间点内保持较高的存活率。
较高的加里浓度显示存活率暂时下降，随后部分恢复且随时间变异性增加。阳性结果包括运动性降低的圆润或半坚持细胞，且在反复亚培养中保持完整。适应性E. gracilis培养物在多种底质类型中表现出可重复的生长，并且较野生型培养物更早稳定，晚期衰退减少较小。
该方案的成功实施使E. gracilis生物质通过外部实验室使用燃烧Dumas方法的粗蛋白含量可测量。适应培养中观察到的蛋白质百分比与野生型培养相若，野生型培养物总蛋白质产量较高，生物量积累增加。该方案使研究人员能够研究尤格林植物生长、氰化物减少、生物质生产及不同木薯环境下的营养潜力。
该方案的关键挑战是维持稳定的温度、一致的培养条件以及均衡的微生物群落，以促进尤格兰菌的生长。未来的研究可以优化和扩大该方案，提高蛋白质产量，并在大规模上测试不同工业应用，利用多样化的木人蜡废弃物流。
Overview
This protocol outlines a multistage adaptation strategy to establish a cassava-tolerant lineage of Euglena gracilis for heterotrophic cultivation in cassava-derived substrates. The method addresses challenges posed by variable composition, cyanogenic compounds, and microbial communities in cassava processing by-products, aiming to enable robust algal growth for bioprocessing and waste valorization.
Key Study Components
Area of Science
- Microbial biotechnology
- Algal cultivation
- Bioprocess engineering
Background
- Cassava processing generates nutrient-rich by-products that are underutilized.
- Challenges include variable substrate composition, presence of cyanogenic compounds, and active microbial communities.
- Euglena gracilis is a versatile microalga with potential for bioprocessing applications.
- Developing cassava-tolerant algal strains could enhance waste valorization.
Purpose of Study
- To develop a reproducible protocol for adapting Euglena gracilis to cassava-derived substrates.
- To assess the tolerance and growth performance of adapted strains in various cassava by-products.
- To enable the use of robust algal lineages in cassava-focused bioprocessing.
Methods Used
- Simultaneous exposure of Euglena cultures to autoclaved and non-autoclaved cassava media.
- Application of a garri concentration gradient (10–30 g/L) to assess tolerance to substrate load and microbial complexity.
- Further adaptation with higher garri concentrations (30–50 g/L).
- Comparative growth assessment of adapted and wild-type strains across five cassava products: garri, dried pulp, pellets, peels, and animal feed.
- Measurement of pH changes and cyanide levels using colorimetric strips.
- Preparation of dried biomass for protein analysis via total nitrogen quantification.
Main Results
- Establishment of a cassava-tolerant Euglena gracilis lineage suitable for heterotrophic cultivation.
- Demonstrated growth consistency of the adapted strain in diverse cassava-derived substrates.
- Effective monitoring of pH and cyanide levels during adaptation and cultivation.
- Protocol enables reliable protein analysis of algal biomass grown on cassava by-products.
Conclusions
- The protocol provides a reproducible framework for adapting microalgae to complex, variable agro-industrial substrates.
- Adapted Euglena gracilis strains show promise for cassava waste valorization and bioprocessing applications.
- This approach supports the development of sustainable biotechnological solutions for agricultural by-product utilization.
Frequently Asked Questions
Executive Industry Relevance
Adapting Euglena gracilis to cassava-based substrates addresses the challenge of utilizing variable, nutrient-rich agro-industrial by-products in bioprocessing pipelines. This method enables robust strain development for heterotrophic cultivation, supporting predictive confidence in substrate tolerance and consistent biomass output. The approach is strategically positioned for portfolio expansion in sustainable bioprocessing and waste valorization initiatives.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables systematic interrogation of algal tolerance to complex, variable substrates.
- Supports biological de-risking by identifying strains resilient to cyanogenic compounds and microbial complexity.
- Facilitates predictive confidence in strain selection for downstream bioprocessing.
Screening & Assay Development
- Establishes validated adaptation protocols for reproducible strain performance assessment.
- Standardizes measurement of pH, cyanide, and protein content for quantitative comparison.
- Prepares adapted lineages for scalable screening across diverse cassava-derived materials.
Translational & Preclinical Research
- Aligns strain adaptation with field-relevant substrate diversity for translational continuity.
- Enables risk-adjusted advancement of robust strains into pilot-scale bioprocessing.
- Provides a framework for evaluating bioprocessing candidates in real-world substrate conditions.
Pipeline & Workflow Integration
This adaptation protocol fits within the early discovery to lead identification continuum for bioprocessing strain development, bridging laboratory adaptation with field-relevant substrate testing.
- Discovery Biology: Supports hypothesis testing on substrate tolerance and metabolic resilience.
- Screening: Delivers reproducible, quantitative outputs for cross-strain and cross-substrate comparison.
- Analytics: Integrates pH, cyanide, and protein measurements for robust data-driven decisions.
- Translational Research: Ensures continuity from adaptation to application in diverse cassava by-products.
- Enterprise Reuse: Provides a reusable protocol for adapting microalgae to other agro-industrial substrates.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence in strain performance and substrate compatibility.
- Operational Value: Standardizes adaptation and measurement workflows for reproducibility and scalability.
- Strategic Value: Enables informed go/no-go decisions for strain advancement in bioprocessing pipelines.
- Portfolio Impact: Supports risk-adjusted prioritization of robust, field-ready algal lineages.
Implementation Considerations
- Requires expertise in algal cultivation and adaptation protocols.
- Needs access to analytical tools for pH, cyanide, and protein quantification.
- Demands cross-team standardization for reproducible adaptation and measurement.
- Must consider substrate variability and microbial complexity in protocol adaptation.
- Practical limitations include the need for safe handling of cyanogenic compounds.
