Name: A Method for Adapting Euglena gracilis to Cassava-Based Substrates for Heterotrophic Cultivation (Video) | JoVE Journal
Uploaded: 2026-06-12T14:40:00.000+00:00
Duration: 12 min 37 s
Description: Learn a multistage adaptation protocol for cultivating Euglena gracilis on cassava-derived substrates. This method enables robust heterotrophic growth for sustainable bioprocessing and agricultural waste valorization.

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5 minutes

The authors thank&#160;Psaltry&#160;International Company Ltd. for supplying cassava-derived materials and for contributing to the definition of applied research&#160;objectives&#160;relevant to industrial cassava processing.&#160;
The authors&#160;acknowledge&#160;Resourced Global Inc. (Lead of the Rockefeller Foundation supported NFAL Program).&#160;
The authors&#160;acknowledge Soci&#233;t&#233; G&#233;n&#233;rale de Surveillance (SGS) for conducting external crude protein analyses using the&#160;Combustion (Dumas)&#160;method.&#160;Additional&#160;laboratory support and methodological feedback were provided by members of the Trent University research community.&#160;
This work was supported in part by partner-supported applied research initiatives.&#160;

The authors declare no competing&#160;financial interests&#160;or conflicts of interest.&#160;

A central determinant of success in this protocol is the balance between cassava substrate concentration and physiological stability during heterotrophic cultivation of&#160;E.&#160;gracilis.&#160;Cassava-derived materials introduce physical heterogeneity, residual particulates, and variable nutrient release kinetics that directly influence growth behavior and culture stability2,3.&#160;Among the protocol steps, substrate preparation and concentration&#160;selection&#160;therefore represent the most sensitive control points. As&#160;demonstrated&#160;by the Representative Results, intermediate substrate concentrations reliably support sustained growth with limited replicate divergence,&#160;whereas&#160;lower concentrations constrain biomass accumulation and higher concentrations increase temporal variability and transient stress responses. These outcomes underscore that maximal substrate loading does not necessarily yield&#160;optimal&#160;or stable cultivation performance when working with heterogeneous agro-industrial materials.&#160;The selected preprocessing steps and substrate concentration ranges were designed to progressively challenge E. gracilis under increasing chemical and microbial complexity. The observed differences in growth stability and viability across these conditions support the validity of this staged approach and confirm that intermediate substrate concentrations provide a balance between nutrient availability and physiological stress.
Culture handling during heterotrophic growth further influences adaptation outcomes. Consistent but gentle mixing under fully dark conditions was critical for&#160;maintaining&#160;contact between&#160;E.&#160;gracilis&#160;cells and suspended substrate material while avoiding excessive shear stress. Insufficient mixing promoted sedimentation and localized nutrient gradients, confounding both growth and viability measurements, particularly in non-autoclaved media12.&#160;These observations align with&#160;previous&#160;reports showing that spatial heterogeneity and uneven substrate exposure can obscure physiological responses in microbial cultivation systems. The protocol therefore emphasizes routine inversion rather than continuous agitation as a practical compromise between homogeneity and culture integrity.&#160;
To accommodate variability inherent to cassava-based substrates, the protocol incorporates multiple troubleshooting and flexibility points. Autoclaving fermented cassava media improved short-term consistency by reducing microbial load and altering substrate structure, particularly during early adaptation stages. In contrast, non-autoclaved media&#160;can&#160;be&#160;used effectively following successful adaptation, provided inoculum density and mixing were sufficient to&#160;maintain&#160;stable growth. This staged approach is consistent with prior studies&#160;demonstrating&#160;that gradual selective pressure enables microbial populations to tolerate increasing environmental complexity over time13,14. Longitudinal monitoring was essential in this context, as transient reductions in growth or viability&#160;frequently&#160;recovered during&#160;subsequent&#160;passages without compromising overall adaptation success.&#160;
Viability assays and morphological monitoring served as rapid diagnostic tools throughout the adaptation process. Trypan Blue exclusion provided a quantitative indicator of physiological robustness, while microscopy-based assessment of motility and&#160;encystment&#160;offered early warning of suboptimal conditions such as excessive substrate loading or delayed acclimation15. Importantly, the protocol emphasizes trends across time rather than single-point measurements, as short-term fluctuations in viability or morphology were not always predictive of long-term culture failure.&#160;This longitudinal perspective proved critical for distinguishing transient stress responses from sustained physiological decline.&#160;Reductions in detectable cyanide levels observed during cultivation suggest partial detoxification of cassava-derived substrates under the conditions described. These reductions may result from a combination of fermentation-associated degradation, volatilization, and microbial activity. While the specific biochemical mechanisms and kinetics of cyanide removal were not directly investigated in this study, these observations support the inclusion of cyanide monitoring as a practical indicator of substrate transformation and culture feasibility during adaptation.
Several limitations of the method should be acknowledged. First, crude protein percentage alone does not fully capture nutritional value or functional protein quality, and interpretation must consider total biomass output alongside compositional metrics16.&#160;Second, the use of minimally processed, heterogeneous substrates&#160;introduce&#160;variability that cannot be eliminated, even with standardized preparation. This challenge is well recognized in agro-industrial bioprocessing17&#160;and reflects real-world constraints rather than experimental shortcomings. Third, while the protocol is&#160;optimized&#160;for laboratory- and pilot-scale cultivation, scale-up beyond bench-scale volumes will require additional control of mixing dynamics, oxygen transfer, and solids handling, as well as consideration of reactor configuration and hydrodynamic conditions, as has been shown for other microalgal and protist systems18.
In comparison to existing&#160;E.&#160;gracilis&#160;cultivation methods that rely on refined sugars or tightly controlled phototrophic systems, this protocol offers a distinct methodological advantage by enabling growth on low-cost cassava-derived substrates under fully heterotrophic conditions. The approach avoids dependence on purified carbon sources19,20&#160;and does not require specialized lighting infrastructure, expanding the operational flexibility of&#160;E.&#160;gracilis&#160;cultivation. This is particularly relevant for applications focused on resource efficiency, waste valorization, and decentralized production in cassava-producing regions.&#160;
The broader significance of this method lies in its applicability to research areas focused on microbial adaptation, sustainable bioprocessing, and biomass valorization. By coupling incremental substrate exposure with simple, low-cost monitoring tools, the protocol provides a reproducible framework for integrating non-traditional substrates into microbial cultivation workflows. Although developed for&#160;E.&#160;gracilis, the underlying principles of staged adaptation, longitudinal performance assessment, and morphology-informed decision-making are broadly transferable to other microalgal and protist systems being explored for circular bioeconomy applications21,22. As such, this protocol serves not only as a cultivation method but also as a generalizable framework for applied microbial adaptation under real-world substrate constraints.&#160;This method was designed with accessibility as a primary goal and is therefore most suitable for laboratories or applied research settings with basic microbiological infrastructure and access to key biological resources, specifically Euglena gracilis cultures and cassava-derived substrates. Additionally, the system offers temporal flexibility, as cassava-based media can be prepared and fermented year-round without seasonal constraints. While this protocol was developed and validated at the laboratory scale, its use of low-cost cassava-derived substrates and minimal processing requirements supports potential scalability to larger cultivation systems.
While this protocol establishes a reproducible framework for adaptation and cultivation, further quantitative analyses of growth kinetics, biomass productivity, and substrate conversion efficiency will be important for optimizing performance in scaled or industrial applications.

Cassava (Manihot esculenta) is a major food and industrial crop across West Africa, generating processed by-products such as garri, dried pulp, peels, and pellets1.
Cassava derived substrates are primarily composed of fermentable carbohydrates and residual nutrients as well as minor protein and fibre content although composition can vary depending on the processing conditions and source material2,3. They remain underutilized due to their heterogeneous composition, presence of cyanogenic glycosides, and the diverse microbial communities that arise during traditional processing and fermentation4. These characteristics present challenges for direct microbial cultivation and limit the integration of cassava by-products into standardized bioprocessing workflows. Despite the abundance of cassava-derived by-products, there is currently a lack of reproducible and bio-sustainable methods for cultivating microbial systems on these chemically complex and microbially active substrates. A cultivation system capable of enriching or detoxifying such a popular food source while operating reliably under these conditions would support more sustainable cassava-processing workflows and circular resource utilization.
Euglena gracilis is a unicellular flagellate within the Euglenozoa5 recognized for its flexible pellicle, dynamic morphology, and reversible encystment capabilities in response to substantial fluctuations in nutrient availability, osmotic pressure, and chemical composition6. Its metabolic adaptability also allows it to synthesize the storage polysaccharide paramylon6 and regulate cellular pH through organic acid metabolism. These metabolic traits, particularly paramylon accumulation, may enhance energy storage and stress tolerance, supporting survival and adaptation under the variable nutrient and chemical conditions characteristic of cassava-derived substrates. These traits enable E. gracilis to persist under chemically variable freshwater conditions, making it a promising candidate for cultivation on heterogenous, non-refined substrates7.
When exposed to chemically complex or microbially active substrates, non-adapted E. gracilis cultures often exhibit stress responses such as increased encystment and reduced motility, whereas adapted populations may maintain more stable morphology and metabolic activity8. Interactions with associated microbial communities may further influence physiological performance during prolonged exposure to complex substrates. These characteristics highlight the relevance of adaptation-based approaches when cultivating E. gracilis on cassava-derived materials9.
To address these challenges, this method employs a staged adaptation strategy designed to gradually acclimate E. gracilis to cassava-derived substrates. The approach incorporates controlled exposure to increasing substrate complexity and microbial activity, allowing selection of populations capable of maintaining viability and growth under heterogeneous cultivation conditions. This design allows E. gracilis to interact with, and potentially derive advantages from, the associated microbiota, and adapt to increasing media concentrations.
Following adaptation, the protocol leads a comparative evaluation of adapted and non-adapted E. gracilis lineages across multiple cassava-derived products. This comparative framework allows assessment of growth consistency, morphological stability, and physiological responses under field-relevant substrate conditions, as the adaptation process gradually prepares E. gracilis to navigate the nutrient variability, chemical stressors, and microbial activity characteristic of cassava by-products.
The method incorporates standardized monitoring of pH and cyanide levels10 during cultivation, as well as preparation of harvested biomass for downstream protein analysis via total nitrogen quantification11, enabling comparison of nutritional potential across substrates and strains. Together, these measurements support evaluation of metabolic stability, substrate interactions, and biomass characteristics during cassava-based cultivation and adaptation to demonstrate that adapted E. gracilis lineage can grow robustly and consistently across multiple cassava by-products.
This method is most suitable for laboratory or applied research settings with access to basic microbiological infrastructure and locally available cassava-derived substrates. Practical considerations include the need for consistent handling of heterogeneous media, access to microscopy for monitoring culture health, and careful control of mixing to avoid shear stress during cultivation.

<table><tbody><tr><td>Autoclave -&lt;br/&gt; 533LS Steam&lt;br/&gt; Sterilizer</td><td>Getinge</td><td>533LS</td><td>Steam sterilization of cassava media.&lt;br/&gt; Sterilize media using liquid cycle.</td></tr><tr><td>Bright-Line&amp;trade;&lt;br/&gt; Hemacytometer&lt;br/&gt; replacement&lt;br/&gt; cover slip</td><td>Millipore Sigma</td><td>BR723015-&lt;br/&gt; 100EA</td><td>Specialized thick coverslips designed for hemocytometer use.</td></tr><tr><td>Cassava dried&lt;br/&gt; pulp</td><td>Psaltry&lt;br/&gt; International</td><td>N/A</td><td>Provided cassava substrate</td></tr><tr><td>Cassava pellets</td><td>Psaltry&lt;br/&gt; International</td><td>N/A</td><td>Provided cassava substrate</td></tr><tr><td>Cassava peels</td><td>Psaltry&lt;br/&gt; International</td><td>N/A</td><td>Provided cassava substrate</td></tr><tr><td>Cassava&lt;br/&gt; substrate (garri)</td><td>Local supplier</td><td>N/A</td><td>Cassava-derived garri&lt;br/&gt; feedstock used as carbon&lt;br/&gt; source; prepared with&lt;br/&gt; deionized water.</td></tr><tr><td>Centrifuge&lt;br/&gt; Thermo&lt;br/&gt; Scientific Sorvall&lt;br/&gt; ST16 Centrifuge</td><td>Thermo&lt;br/&gt; Scientific Sorvall&lt;br/&gt; ST16 Centrifuge</td><td>75004241</td><td>Capable of &amp;ge;3,500 &amp;times; g, used for harvesting biomass.</td></tr><tr><td>Centrifuge tubes&lt;br/&gt; E.g. Axygen (A&lt;br/&gt; Corning Brand)</td><td>Corning</td><td>MCT-150-C-S</td><td>Sterile, minimum 1.5 mL volume</td></tr><tr><td>Clean water&lt;br/&gt; E.g. UltraPure&amp;trade;&lt;br/&gt; DNase/RNase-&lt;br/&gt; Free Distilled&lt;br/&gt; Water</td><td>ThermoFisher&lt;br/&gt; Scientific</td><td>10977035</td><td>Deionized or distilled. Used for media preparation</td></tr><tr><td>Compound light&lt;br/&gt; microscope</td><td>Olympus</td><td>N/A</td><td>Standard compound microscope suitable for routine cell observation and counting.</td></tr><tr><td>Cyanide Test&lt;br/&gt; Strips,&lt;br/&gt; colorimetric.&lt;br/&gt; CN&lt;sup&gt;-&lt;/sup&gt; 10-30 mg/L</td><td>MQuant</td><td>110044</td><td>Semi-quantitative cyanide detection. Dispose as hazardous waste</td></tr><tr><td>Sterile Standard&lt;br/&gt; Weighing Boat</td><td>Fisher Scientific</td><td>01-549-750</td><td>Used for weighing cassava substrates and dry biomass.</td></tr><tr><td>Euglena gracilis&lt;br/&gt; (adapted&lt;br/&gt; lineage)</td><td>N/A</td><td>N/A</td><td>Cassava adapted strain generated in this study</td></tr><tr><td>Euglena gracilis&lt;br/&gt; (Wild-type)</td><td>N/A</td><td>N/A</td><td>Non-adapted reference strain. Maintain the same standard conditions.</td></tr><tr><td>Euglena growth&lt;br/&gt; medium&lt;br/&gt; components</td><td>Various</td><td>N/A</td><td>Basal Euglena medium prepared according to laboratory standard formulations.</td></tr><tr><td>FreeZone 6 Liter&lt;br/&gt; Console Freeze&lt;br/&gt; Dryer</td><td>Labconco</td><td>70061xxxx</td><td>Used for biomass dehydration</td></tr><tr><td>Hemocytometer&lt;br/&gt; with Standard&lt;br/&gt; Lines</td><td>Weber&lt;br/&gt; Scientific</td><td>NC1587539</td><td>Used for manual cell counting.</td></tr><tr><td>Magnetic stirrer&lt;br/&gt; with hot plate</td><td>IKA</td><td>N/A</td><td>Optional; improves consistency during media preparation and substrate suspension.</td></tr><tr><td>Micropipettes&lt;br/&gt; (P20, P200,&lt;br/&gt; P1000)</td><td>Eppendorf</td><td>N/A</td><td>Adjustable volume pipettes used for aseptic liquid transfers.</td></tr><tr><td>Mini-centrifuge</td><td>Eppendorf</td><td>N/A</td><td>Optional; facilitates rapid pelleting during biomass harvesting.</td></tr><tr><td>Parafilm</td><td>Bemis</td><td>PM996</td><td>Used for temporary sealing of flasks or plates when vented caps are unavailable.</td></tr><tr><td>pH indicator&lt;br/&gt; strips</td><td>Fisher Scientific</td><td>13-640-520</td><td>Alternative to pH meter for rapid qualitative assessment of culture media.</td></tr><tr><td>pH meter with&lt;br/&gt; probe&lt;br/&gt; Thermo&lt;br/&gt; Scientific Orion&lt;br/&gt; Star A211&lt;br/&gt; Benchtop pH&lt;br/&gt; Meter</td><td>Thermo&lt;br/&gt; Scientific Orion&lt;br/&gt; Star A211&lt;br/&gt; Benchtop pH&lt;br/&gt; Meter</td><td>13-645-519</td><td>Calibrated before use. For measuring the pH of the culture supernatant</td></tr><tr><td>Refrigerated&lt;br/&gt; storage (4&amp;deg;C)</td><td>Frigidaire</td><td>FRT18S6JW7</td><td>Short term storage of media</td></tr><tr><td>Shaking&lt;br/&gt; incubator (dark)</td><td>Thermolyne</td><td>M49235</td><td>Orbital shaking incubator capable of 100&amp;ndash;150 RPM, temperature range 24&amp;ndash;28 &amp;deg;C; used under dark conditions to support heterotrophic Euglena growth.</td></tr><tr><td>Spray bottle&lt;br/&gt; (32oz)</td><td>Boardwalk</td><td>BWK03010</td><td>Used for benchtop sterilization and routine aseptic handling (70% ethanol solution)</td></tr><tr><td>Spreadsheet&lt;br/&gt; software</td><td>Microsoft Excel</td><td>N/A</td><td>Data collection, organization and analysis</td></tr><tr><td>Sterile pipette&lt;br/&gt; tips</td><td>Eppendorf</td><td>N/A</td><td>Sterile, disposable tips compatible with P20, P200, and P1000 pipettes.</td></tr><tr><td>Sterile&lt;br/&gt; serological&lt;br/&gt; pipettes (5, 10,&lt;br/&gt; 25 mL)</td><td>Corning</td><td>N/A</td><td>Individually wrapped sterile pipettes used for culture transfers and sampling.</td></tr><tr><td>Trypan Blue&lt;br/&gt; solution&lt;br/&gt; 0.4%, liquid,&lt;br/&gt; sterile-filtered,&lt;br/&gt; suitable for cell&lt;br/&gt; culture</td><td>Sigma-Aldrich</td><td>T8154</td><td>Vital dye used for cell viability assessment via Trypan Blue exclusion assay.</td></tr><tr><td>Tubes for sample&lt;br/&gt; collection (50&lt;br/&gt; mL)</td><td>Falcon</td><td>14-432-22</td><td>For pH and cyanide sampling</td></tr><tr><td>Vent-cap&lt;br/&gt; Erlenmeyer&lt;br/&gt; flasks (250&amp;ndash;500&lt;br/&gt; mL)</td><td>Corning&amp;reg; 250 mL Polycarbonate Erlenmeyer Flask with Vent Cap</td><td>431144</td><td>Polycarbonate Erlenmeyer flasks with vented caps; autoclavable; used for both sterile and non-sterile liquid cultures.</td></tr><tr><td>Waste containers</td><td>Various - in compliance with set laboratory standards</td><td>N/A</td><td>Autoclave bags and sharps or glass waste containers for biosafety-compliant disposal.</td></tr></tbody></table>

a method for adapting euglena gracilis to cassava-based substrates for heterotrophic cultivation

A Method for Adapting Euglena gracilis to Cassava-Based Substrates for Heterotrophic Cultivation

Cassava processing generates nutrient-rich by-products that remain underutilized due to variable composition, cyanogenic compounds, and active microbial communities. This protocol presents a multistage adaptation strategy for establishing a cassava-tolerant lineage of Euglena gracilis suitable for heterotrophic cultivation in cassava-derived substrates. The method begins with simultaneous exposure of Euglena cultures to autoclaved and non-autoclaved cassava media and to a garri concentration gradient of 10&#8211;30 g/L to assess tolerance to microbial complexity and substrate load. Cultures are then subjected to a higher concentration gradient of 30&#8211;50 g/L to evaluate performance with denser substrates. The resulting lineage is compared with a non-adapted wild-type strain across five cassava products; garri, dried pulp, pellets, peels, and animal feed to assess growth consistency in field-relevant materials. The protocol includes measurement of pH changes, cyanide levels using colorimetric strips, and preparation of dried biomass for protein analysis based on total nitrogen quantification. Together, these procedures provide a reproducible framework for adapting Euglena gracilis to cassava-derived substrates and for evaluating strain performance across diverse cassava products. This method enables the development of robust algal lineages for use in cassava-focused bioprocessing and waste-valorization applications.

The following representative results illustrate the expected outcomes when the described protocol is applied successfully, as well as the range of outcomes observed under suboptimal conditions. Representative data corresponding to these outcomes are shown in Figure 1, Figure 2, Figure 3, Figure 4, Figure 5, Figure 6, Figure 7. Together, these results provide practical guidance for interpreting growth dynamics, viability, morphology, and protein outcomes when adapting E. gracilis to cassava-based substrates under heterotrophic, fully dark cultivation conditions. Moreover, these results validate key biological outcomes of the protocol, including successful adaptation, sustained growth, high viability, stable morphology, consistent performance across substrates, and measurable protein production.
Throughout the experiments, three replicates per treatment (all types of cassava media) were utilized. We acknowledge that the number of replicates is up to the discretion and logistical limitations of any group following the protocols above. Successful application of this protocol results in sustained heterotrophic growth of E. gracilis in garri-based media under fully dark conditions. Positive outcomes are characterized by smooth, sigmoidal growth trajectories, limited divergence among replicates, and stable late-phase cell densities. Morphological traits that signified positive outcomes and were consistently observed in adapted populations included cell roundness and thick cell membranes. In contrast, unhealthy collapsed cells typically appear irregular, fragmented, or structurally compromised and are associated with reduced viability, distinguishing them from intact rounded or semi-encysted forms observed in adapted cultures. For reference, representative images of wild-type E. gracilis exhibiting characteristic elongated morphology are included (Figure 8) to distinguish baseline cellular structure from adapted or altered forms. Cultures grown at 30 g L-1 garri exhibited higher integrated growth, as measured by area under the growth curve (AUC), compared with lower concentrations, suggesting more efficient utilization of the cassava substrate and successful physiological adaptation (Figure 1).
Suboptimal outcomes are commonly observed at lower garri concentrations (10–20 g L-1), where growth is detectable but reduced in magnitude and more variable across replicates. At higher concentrations (40–50 g L-1), cultures may reach elevated peak cell densities; however, these conditions frequently display delayed stabilization or increased temporal variability (Figure 2).
In successful experiments, cell viability remains high throughout the culture period, with only minor mid-experiment fluctuations. Trypan Blue exclusion assays indicate that cultures maintained at 30 g L-1 garri retain a high proportion of viable (unstained) cells relative to non-viable (blue-stained) cells across sampled time points (Figure 3). Deviations from this pattern, including an increased proportion of stained cells, reduced motility, or shifts toward rounded or collapsed morphologies, may indicate suboptimal conditions and serve as early triggers for troubleshooting. Viability and morphological assessments were based on consistent observational criteria, including cell integrity, directional motility, and the relative prevalence of elongated versus rounded or collapsed forms.
Suboptimal outcomes are characterized by transient reductions in viability, most commonly observed at higher garri concentrations. Partial recovery may be observed at later time points; however, these conditions display increased variability in viability across the culture period (Figure 4). These growth patterns show that intermediate substrate concentrations (e.g., 30 g L-1) provide a balance between nutrient availability and stress, resulting in more stable growth. These trends reflect the impact of preprocessing conditions and staged adaptation on culture performance, as gradual exposure to increasing substrate complexity supports improved physiological stability and reproducible growth behavior. Lower concentrations limit biomass production, while higher concentrations lead to greater variability and delayed stabilization.
Morphological assessment provides a qualitative indicator of adaptation status. Positive outcomes include the emergence and persistence of rounded or semi-encysted cells with reduced motility that remain intact across repeated subculturing (Figure 5).
By contrast, suboptimal cultures, most associated with wild-type strains or early-stage adaptation, exhibit abrupt transitions from elongated, motile cells to irregular or collapsed morphologies that coincide with reduced growth stability.
When the protocol is applied to additional cassava-derived substrates, including pulp, pellets, and peels, adapted E. gracilis cultures display reproducible growth across multiple substrate types. Positive outcomes are characterized by earlier stabilization of growth curves and reduced late-stage decline relative to wild-type cultures (Figure 6).
Suboptimal outcomes include delayed growth initiation or increased replicate variability, particularly in substrates with greater physical heterogeneity.
Successful implementation of the protocol yields E. gracilis biomass with measurable crude protein content, as quantified by external laboratory analysis using the Combustion (Dumas) method. Protein percentages observed in adapted cultures are comparable to those measured in wild-type cultures, with higher total protein output corresponding to increased biomass accumulation (Figure 7).
Monitoring of media chemistry supported culture–substrate interactions during adaptation and validated the inclusion of pH and cyanide measurements within the protocol. pH values showed gradual shifts over time, indicating metabolic activity and substrate utilization, with more stable trends in adapted cultures. Cyanide measurements further supported this, as adapted cultures showed reductions in detectable levels, while wild-type cultures remained more variable. Together, these results demonstrate that these monitoring steps effectively capture changes in media chemistry associated with adaptation and support interpretation of culture performance. Although the underlying mechanisms were not investigated, this represents an important area for future research, particularly for industrial-scale applications.
The long-term maintenance and consolidation workflow supported continued viability of the adapted Euglena gracilis lineage over extended culture periods. Cultures maintained through repeated passaging consistently retained motility, characteristic morphology, and stable growth behavior across maintenance cycles. Although long-term performance was not quantitatively assessed, these observations provide representative evidence that the adapted lineage can be reliably maintained under the specified conditions.
Together, these results confirm that the adaptation protocol supports robust growth, viability, and biomass production of E. gracilis across cassava-based substrates, with consistent performance and reduced cyanide levels over time.
All figures and tables are provided as separate files and are not embedded within the manuscript document. Figure and table placement preferences are indicated within the manuscript text using bracketed notes (e.g., (Figure 1)). Unless otherwise specified, all figures and tables are intended to appear below the Representative Results section in the final publication.
All figures are submitted as individual files, with multipaneled figures provided as a single combined image. Microscopy images include scale bars in each panel. Data figures include labeled axes with defined measurement units, and error bars are specified in the corresponding figure legends.
All tables are submitted as individual .xls or .xlsx files and contain raw or summarized numerical data without decorative formatting.
<img alt="Euglena growth dynamics chart; cell density vs. days; autoclaved vs. non-autoclaved cultures." class="xfigimg" src="/files/ftp_upload/70741/70741fig1.jpg" data-altupdatedat="1784001604000" data-alt="Figure 1"> 
Figure 1: Growth dynamics of E. gracilis cultured at 10, 20, and 30 gL-1 garri under fully dark conditions. Mean cell densities are shown over time for each substrate concentration. Error bars represent SEM across biological replicates. <a href="https://www.jove.com/files/ftp_upload/70741/70741fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Cell density vs. time graph; analysis of growth across garri concentrations; data comparison." class="xfigimg" src="/files/ftp_upload/70741/70741fig2.jpg" data-altupdatedat="1784001604000" data-alt="Figure 2"> 
Figure 2: Growth performance of E. gracilis at elevated garri concentrations (30–50 g L-1). Cell density trajectories illustrate differences in peak density, stabilization timing, and variability across higher substrate concentrations. <a href="https://www.jove.com/files/ftp_upload/70741/70741fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Cell viability graph of autoclaved agar in Euglena line selection, showing viability over days." class="xfigimg" src="/files/ftp_upload/70741/70741fig3.jpg" data-altupdatedat="1784001604000" data-alt="Figure 3"> 
Figure 3: Cell viability trends of E. gracilis cultured at 30 gL-1 garri measured by Trypan Blue exclusion. The proportion of viable cells is shown across sampled time points. <a href="https://www.jove.com/files/ftp_upload/70741/70741fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Cell viability dynamics across garri concentrations; line graph showing stress and recovery over time." class="xfigimg" src="/files/ftp_upload/70741/70741fig4.jpg" data-altupdatedat="1784001604000" data-alt="Figure 4"> 
Figure 4: Viability fluctuations of E. gracilis cultured at higher garri concentrations. Transient changes in cell viability are shown across the experimental period. <a href="https://www.jove.com/files/ftp_upload/70741/70741fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Microscopic image of parasitic examination showing various stages of nematodes in sample analysis." class="xfigimg" src="/files/ftp_upload/70741/70741fig5.jpg" data-altupdatedat="1784001604000" data-alt="Figure 5"> 
Figure 5: Representative morphology of adapted E. gracilis populations. Representative inverted light microscopy images show rounded and semi-encysted cells observed following adaptation. Scale bar = XX µm. <a href="https://www.jove.com/files/ftp_upload/70741/70741fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Growth trends diagram; cell density vs. calendar day; GA/WT strains; statistical analysis; GAM lines." class="xfigimg" src="/files/ftp_upload/70741/70741fig6.jpg" data-altupdatedat="1784001604000" data-alt="Figure 6"> 
Figure 6: Growth performance of adapted and wild-type E. gracilis across cassava-derived substrates. Growth trajectories are shown for adapted and wild-type cultures cultivated on multiple cassava products. <a href="https://www.jove.com/files/ftp_upload/70741/70741fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Protein concentration analysis; graph of total protein vs. condition; data comparison; experimental results." class="xfigimg" src="/files/ftp_upload/70741/70741fig7.jpg" data-altupdatedat="1784001604000" data-alt="Figure 7"> 
Figure 7: Crude protein content and total protein output of cassava-grown E. gracilis biomass. Protein content was quantified by external analysis using the SGS Combustion (Dumas) method. Total protein output is shown alongside biomass accumulation. <a href="https://www.jove.com/files/ftp_upload/70741/70741fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Microscope image of plankton; cellular structure; optical study; aquatic biology research." class="xfigimg" src="/files/ftp_upload/70741/70741fig8.jpg" data-altupdatedat="1784001604000" data-alt="Figure 8"> 
Figure 8: Representative morphology of wild-type E. gracilis under standard conditions (40X magnification). Representative image of healthy, non-adapted Euglena gracilis showing characteristic elongated morphology under light microscopy. <a href="https://www.jove.com/files/ftp_upload/70741/70741fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

This protocol describes the stepwise method for adapting&#160;Euglena&#160;gracilis&#160;to cassava-based substrates and the&#160;subsequent&#160;evaluation of growth, viability, and biomass production across multiple cassava products under heterotrophic cultivation conditions.&#160;&#160;

Cassava processing generates nutrient-rich by-products that remain underutilized due to variable composition, cyanogenic compounds, and active microbial ...

Our research focuses on adapting new Euglena gracilis leaves to cassava waste and studying growth, cyanide reduction, and protein-rich biomass production. This protocol can be applied in sustainable cassava processing, agricultural waste valorization, feed production, and low-cost biotechnology systems. To begin, label sterile 500-milliliter vented cap flasks with the substrate type as garri.Mark the appropriate target concentration on the flask. Label the flask with the replicate identification and planned sterilization condition as autoclaved or non-autoclaved. Place a weighboat on the analytical balance.After pressing tare, weigh the garri to match the final working volume. Aseptically add the weighed garri into a sterile 500-milliliter flask containing 300 milliliters of deionized or distilled water. Reapply the vented cap to each flask.Mix each suspension for three minutes until the mixture is homogenized. If magnetic stirring is used, place the flask on a stir plate and set the speed to 200G to form a shallow vortex without splashing. Allow the suspension to hydrate undisturbed for 10 minutes at 25 degrees Celsius.Tighten the vent cap immediately prior to fermentation and swirl the flask by hand five times to gently homogenize the mixture and re-suspend the settled material. To prepare additional cassava product media, label the flasks and weigh the substrate as previously demonstrated to achieve 30 grams per liter based on the working volume. Aseptically add the weighed substrate into a sterile flask containing 300 milliliters of deionized or distilled water.Reapply the vented cap to each flask. Hydrate and mix the suspension as previously demonstrated. Confirm that flasks are fitted with a sterile gas-permeable vented cap to permit gas exchange while minimizing environmental contamination.Place the flasks upright at 25 degrees Celsius in a designated area away from direct sunlight and high traffic airflow and incubate the flasks without agitation for a minimum of 72 hours. Record the fermentation start date and time for each flask and condition. Confirm that each 500-milliliter flask contains no more than 300 milliliters of liquid following fermentation to prevent boilover during sterilization.Once the vented cap is loosened to a finger tight position, apply autoclave indicator tape to the flask. After loading the flasks into an autoclave-safe tray, place the tray into the autoclave and select the P13 liquid cycle. Set the sterilization parameters to 121 degrees Celsius for 30 minutes for liquid sterilization.Start the autoclave cycle. After the cycle completes, allow the autoclave chamber pressure to return to atmospheric pressure and open the autoclave door slowly. Using heat-resistant gloves, remove the flasks and allow them to cool to room temperature.Store the cooled flasks overnight at room temperature prior to inoculation. Use the fermented cassava media directly without autoclaving to preserve native microbial communities. Inoculate the non-autoclaved media immediately after the 72-hour fermentation period to minimize unintended environmental contamination.Maintain consistent handling across replicates by using the same type 500-milliliter vented cap flasks, the same working volume, and the same fermentation duration for all substrate conditions. Inoculate Euglena gracilis into fresh heterotrophic nutrient medium. Incubate the cultures in complete darkness at 28 degrees Celsius with continuous orbital shaking at 120G until the cultures reach mid-logarithmic growth.Using a compound light microscope in brightfield mode, examine the cultures at 100x to 400x total magnification and confirm active motility, elongated cell morphology, and low levels of insistment. Exclude cultures exhibiting excessive cellular debris, abnormal morphology, or elevated insistment prior to adaptation experiments. After gently homogenizing the starter cultures by tightening the vent cap, swirl the flasks by hand for 15 seconds to ensure uniform cell distribution without inducing sheer stress.Using a Neubauer hemocytometer, determine the cell density. Once the culture is mixed by swirling the flask for 15 seconds, pipette one milliliter of culture into a sterile 1.5 milliliter microcentrifuge tube. Clean the hemocytometer and cover slip with 70%ethanol and allow the hemocytometer and cover slip to air dry.Pipette 10 microliters of culture onto the two grids on the hemocytometer and place the cover slip onto the wet sample. Using brightfield microscopy with a 10x objective, focus on the grid and count the cells within a defined set of squares on the large grid by applying the standard boundary rules. Repeat the cell counts on the second chamber and calculate the mean cells per milliliter and cell density.Adjust the inoculation volumes to achieve a final inoculum of 5%volume by volume across all experimental media. Prepare separate inocula for each treatment condition to maintain consistent initial cell densities between experimental groups. Add the prepared E.gracilis inoculum to autoclaved and non-autoclaved cassava media prepared at final garri concentrations of 10, 20, and 30 grams per liter.Use equal inoculation volumes across all treatment conditions to standardize the initial cell density. After tightening the vented cap, gently swirl the flask in a circular motion for 15 seconds to distribute the cells. Incubate the cultures in complete darkness at 25 degrees Celsius to initiate heterotrophic growth.Minimize agitation of the non-autoclaved media to preserve the native microbial community structure and substrate stratification. Mix all cultures once daily using gentle manual swirling for 15 seconds to maintain contact between E.gracilis cells and suspended cassava material. Using a sterile pipette tip, every 48 hours, aseptically collect one milliliter samples from each culture.Tighten the vented cap and gently swirl the culture flask for 15 seconds prior to sampling to ensure even distribution of cells and suspended material. After transferring an aliquot of culture into a sterile 1.5 milliliter microcentrifuge tube, dilute the sample with deionized water as needed to improve visualization of individual cells. Prepare a wet mount microscopy slide by pipetting 10 microliters of the diluted sample onto a clean microscope slide and covering the sample with a cover slip.Using a compound light microscope in brightfield mode, examine the samples at 100x to 400x total magnification and assess cell motility, morphology, and the frequency of insistment as indicators of adaptation status. Every seven days, aseptically transfer 20%volume by volume of each culture into freshly prepared cassava media with the same garri concentration and sterilization conditions. Maintain consistent transfer volumes between passages and repeat serial passaging until the cultures exhibit consistent growth, sustained motility, and low insistment frequency in 30 grams per liter cassava media.Following high concentration cassava adaptation and comparative growth analysis, use colorimetric strips to assess cyanide levels and observe the developed color. After centrifugation, harvest biomass for downstream analysis. Under fully dark cultivation conditions, cultures grown at 30 grams per liter of garri exhibited higher integrated growth than lower concentrations.Lower garri concentrations of 10 to 20 grams per liter showed detectable but reduced and more variable growth. Higher concentrations of 40 to 50 grams per liter reached elevated peak cell densities but showed delayed stabilization and greater variability. Trypan blue exclusion assays indicated that cultures maintained at 30 grams per liter garri retained high viability across sampled time points.Higher garri concentrations showed transient reductions in viability followed by partial recovery with increased variability over time. Positive outcomes included rounded or semi-insisted cells with reduced motility that remained intact across repeated subculturing. Adapted E.gracilis cultures displayed reproducible growth across multiple substrate types and showed earlier stabilization with reduced late-stage decline relative to wild-type cultures.Successful implementation of the protocol yielded E.gracilis biomass with measurable crude protein content as quantified by external laboratory analysis using the combustion Dumas method. Protein percentages observed in adapted cultures were comparable to those measured in wild-type cultures with higher total protein output corresponding to increased biomass accumulation. This protocol allows researchers to study Euglena growth, cyanide reduction, biomass production, and nutritional potential in different cassava-based environments.The critical challenge in this protocol is maintaining a stable temperature, consistent culture conditions, and a balanced microbial community for Euglena growth. Future studies can optimize and scale up this protocol, improve protein yield, and test different industrial applications using diverse cassava waste streams on a large scale.

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Learn a multistage adaptation protocol for cultivating Euglena gracilis on cassava-derived substrates. This method enables robust heterotrophic growth for sustainable bioprocessing and agricultural waste valorization.

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