Technical Note: Disruption Methods For Efficient Algal Sample Processing

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Why Algae

Algae play crucial roles in ecological, environmental, and biotechnological applications. The study of algae is necessary for a wide range of applications, spanning all sectors of the economy.

Ecological: Algae are vital for global carbon fixations, particularly in marine environments. Algae like phytoplankton are primary carbon fixers and contribute to the biological carbon pump and have an immense impact on carbon flux (Laber et al., 2018). Algae also provide oxygen and nutrients by their role in sustaining marine food webs.

Environmental: Environmental remediation can be achieved by implementing algae to mitigate climate change by capturing carbon dioxide for the atmosphere and water and improving the quality of the air and water. (Laber et al., 2018).

Biotechnology and Biofuels: Oil rich species of Algae have been studied as promising sources for biofuel production. Oleaginous microorganisms are seen as promising sources for biofuel production and could offer an eco-friendly alternative to fossil fuels (Kosa et al., 2018).

Efficient disruption of algal cells is essential for downstream applications such as nucleic acid extraction, protein isolation, and metabolomic studies. Algae have tough cell walls that require mechanical disruption methods to ensure complete lysis. This protocol outlines bead-beating methods for disrupting algae, based on previous studies using zirconium and glass beads for microbial and planktonic organisms. The selection of bead type and size depends on the algal species and desired application.

Importance of Efficient Disruption

The success of nucleic acid extraction relies heavily on the efficient release of DNA and RNA from cells while minimizing degradation or loss. Incomplete lysis can lead to poor yields, skewed community representation, and compromised integrity, which ultimately impacts the reliability of subsequent analyses. The disruption of algal samples often involves mechanical methods, including bead-beating, which employs physical force to break open cell walls.

Bead beating: A Method for Disruption

Bead-beating has become a widely adopted mechanical disruption technique due to its robustness, efficiency, and versatility. The method involves agitating samples in tubes containing beads that collide with the sample, physically disrupting them. The size and composition of the beads play a significant role in the effectiveness of the process, with smaller beads offering higher shear forces and larger beads providing more force for tougher, more resistant cell types (Weigel et al., 2021). This can be coupled with a chemical or enzymatic process to amplify the forces and disrupt even the most difficult sample types.

OPS Diagnostics offers a wide range of bead types and sizes tailored to various sample types, ensuring optimal lysis and recovery of nucleic acids. The GenoGrinder is commonly used for bead-beating, providing controlled agitation speeds and uniform mixing, which is essential for maximizing disruption efficiency (Zoqratt et al., 2020). Other machines such as the Retch/ Tissuelyser can be used as well, however their arcing motion limits the effectiveness of their bead beating. To ensure uniform disruption it may be necessary to stop the machine part way through the disruption and turn the holder for more consistent results.

Bead Selection for Algal Samples

The choice of bead type and size is critical for effective tissue homogenization in various biological samples. Different algal taxa require specific beads to optimize DNA or RNA extraction while minimizing degradation. Different types of microbial and planktonic samples require specific bead materials and sizes for optimal processing. Table 1 provides recommendations for bead types based on different algal sample sources.

  • Arctic phototrophs, such as Micromonas pusilla and Phaeocystis pouchetii, along with Nitrospina, are best processed using 200 µm molecular biology grade zirconium beads, as recommended by Vader et al. (2014) and Yepsen et al. (2018).
  • For eukaryotic plankton and sponge-associated Betaproteobacterium (Crambe crambe), 200 µm low protein binding zirconium beads are suggested based on studies by John et al. (2007, 2009) and Croué et al. (2013). When working with Emiliania huxleyi blooms, a combination of 100 µm and 400 µm diameter beads has been found effective, as indicated by Thamatrakoln et al. (2018) and Laber et al. (2018).
  • Microbial plankton samples are typically processed using 400 µm zirconium beads, as recommended by Joglar et al. (2021) and Siddiqui (2014). Lastly, for oleaginous microorganisms such as Crypthecodinium cohnii, 800 µm acid-washed glass beads have been identified as the most suitable choice, according to Kosa et al. (2018).
Table 1: Recommended beads for several types of algae. 400 µm zirconium beads
Samples Recommended Beads References
Arctic phototrophs
(Micromonas pusilla,
Phaeocystis pouchetii), Nitrospina 		200 µm molecular biology
grade zirconium beads		 Vader et al. (2014)
Yepsen et al. (2018)
Eukaryotic plankton,
sponge-associated
Betaproteobacterium
(Crambe crambe)		 200 µm low protein
binding zirconium beads 		John et al. (2007)
John et al. (2009)
Croué et al. (2013)
Emiliania huxleyi blooms 100 µm and 400 µm
zirconium beads		 Thamatrakoln et al. (2018)
Laber et al. (2018)
Microbial plankton Joglar et al. (2021)
Siddiqui, M. N. (2014).
Oleaginous microorganisms
(Crypthecodinium cohnii)		 800 µm acid washed
silica beads 		Kosa et al. (2018)


Protocol for Bead-Beating Disruption

Materials

Methods

  1. Sample Preparation
    • Harvest algae by centrifugation at 5,000 × g for 10 minutes at 4°C.
    • Remove the supernatant and suspend the pellet in an appropriate lysis buffer.
    • Transfer the suspended sample to a bead-beating tube.
  2. Bead Addition
    • Select beads based on the algal species (Table 1). The choice of bead size affects disruption efficiency and yield; optimization may be required for specific algae. For general algal applications use 200 µm zirconium bead.
  3. Cell Disruption
    • Secure tubes in the bead beater and process for 30-60 seconds at high speed (4-6 m/s).
    • Incubate on ice for 1 minute between cycles to prevent overheating.
    • Repeat for 3-5 cycles until the lysate appears homogenized. Over-beating may cause excessive heat buildup, leading to RNA degradation. Keep samples on ice when necessary.
    • Alternative disruption methods (e.g., sonication, enzymatic lysis) can be tested if bead beating is ineffective for certain species
  4. Lysate Recovery
    • Centrifuge the disrupted sample at 12,000 × g for 5 minutes at 4°C to pellet debris.
    • Carefully transfer the supernatant to a fresh tube for downstream analysis (e.g., DNA, RNA, or protein extraction).
  5. Storage and Further Processing
    • Store extracted material at -80°C for long-term storage or proceed immediately with nucleic acid/protein purification

Considerations and Challenges

While bead-beating is a highly effective method for disrupting algal samples, there are a few important considerations:

  • Sample Type Variability: Algal samples can be difficult to work with due to their diverse forms and growth conditions. Additionally, handling delicate algae species can present challenges due to their insensitivity to laboratory conditions (Thamatrakoln et al., 2018). Different sample types may require optimization of bead sizes, disruption time, and buffer conditions to achieve the best results.
  • RNA Preservation: For RNA-based analyses, it is essential to perform bead-beating on ice and use RNA stabilization buffers to prevent degradation during the lysis process (Di Rienzi et al., 2021).
  • Bead Selection: The bead type and size should match the algal sample being studied. Smaller beads are more effective for fragile cells, while larger beads provide better disruption for tougher cells.
  • Homogenizer Selection: The motion of the homogenizer can hinder homogenization, and the bead selection should account for the machine.
  • Sample Contamination: Algal samples are often contaminated by microbial organisms, complicating the isolation of pure algae cultures. Especially true in field samples, where microbial communities are diverse (John et al., 2007; Yepsen et al., 2018).

Discussion

As a method, bead-beating is an invaluable tool for the efficient disruption of algal samples. It enables the release of high-quality nucleic acids for downstream molecular analyses. By selecting the appropriate beads for the sample type and optimizing processing conditions, researchers can ensure consistent and reproducible results. This method is widely applicable to various research areas, including taxonomy, systematics, genetics, and molecular biology of algae, and it plays a pivotal role in advancing research in phycology.

Algal samples are complex and difficult to work with because of their various forms. Algae are highly sensitive to environmental conditions such as temperature, nutrient availability, and light. Studies demonstrate varying environmental conditions affect algal interactions, making laboratory testing difficult. However, understanding and overcoming the hurdles these organisms present researchers, will only serve to increase the tools available to humanity for solving the problems presented in front of us.


References

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  2. Di Rienzi, S. C., Johnson, E. L., Waters, J. L., Kennedy, E. A., Jacobson, J., Lawrence, P., Wang, D. H., Worgall, T. S., Brenna, J. T., & Ley, R. E. (2021). The microbiome affects liver sphingolipids and plasma fatty acids in a murine model of the Western diet based on soybean oil. Journal of Nutritional Biochemistry, 97, 108808. https://doi.org/10.1016/j.jnutbio.2021.108808
  3. Joglar, V., Pontiller, B., Martínez-García, S., et al. (2021). Microbial plankton community structure and function responses to vitamin B12 and B1 amendments in an upwelling system. Applied and Environmental Microbiology, 87(21), e01056-21. https://doi.org/10.1128/AEM.01056-21
  4. John, D. E., Patterson, S. S., & Paul, J. H. (2007). Phytoplankton-Group specific Quantitative Polymerase chain reaction assays for RUBISCO mRNA transcripts in seawater. Marine Biotechnology, 9(6), 747-759. https://doi.org/10.1007/s10126-007-9027-z
  5. John, D. E., Zielinski, B. L., & Paul, J. H. (2009). Creation of a pilot metatranscriptome library from eukaryotic plankton of a eutrophic bay (Tampa Bay, Florida). Limnology and Oceanography Methods, 7(3), 249-259. https://doi.org/10.4319/lom.2009.7.249
  6. Kosa, G., Vuoristo, K. S., Horn, S. J., Zimmermann, B., Afseth, N. K., Kohler, A., & Shapaval, V. (2018). Assessment of the scalability of a microtiter plate system for screening of oleaginous microorganisms. Applied Microbiology and Biotechnology, 102(11), 4915-4925. https://doi.org/10.1007/s00253-018-8920-x
  7. Laber, C. P., Hunter, J. E., Carvalho, F., Collins, J. R., et al. (2018). Coccolithovirus facilitation of carbon export in the North Atlantic. Nature Microbiology. https://doi.org/10.1038/s41564-018-0128-
  8. Siddiqui, M. N. (2014). Oxidative stress in algae: Method development and effects of temperature on antioxidant nuclear signaling compounds (Order No. 1566112). Available from ProQuest Dissertations & Theses Global. (1619385713). Retrieved from https://www.proquest.com/dissertations-theses/oxidative-stress-algae-method-development-effects/docview/1619385713/se-2
  9. Thamatrakoln, K., Talmy, D., Haramaty, L., Maniscalco, C., Latham, J. R., Knowles, B., Natale, F., Coolen, M. J. L., Follows, M. J., & Bidle, K. D. (2018). Light regulation of coccolithophore host-virus interactions. New Phytologist, 221(3), 1289-1302. https://doi.org/10.1111/nph.15459
  10. Vader, A., Marquardt, M., Meshram, A. R., & Gabrielsen, T. M. (2014). Key Arctic phototrophs are widespread in the polar night. Polar Biology, 38(1), 13-21. https://doi.org/10.1007/s00300-014-1570-2
  11. Weigel K.M., Olson A.M., Cangelosi G.A. Steady-State pre-rRNA analysis to investigate the functional microbiome. (2021) Current Protocol. 1(7):e209. https://currentprotocols.onlinelibrary.wiley.com/doi/10.1002/cpz1.209
  12. Yepsen, D. V., Levipan, H. A., & Molina, V. (2018). Nitrospina bacteria in a rocky intertidal habitat (Quintay Bay, central Chile). Microbiology Open, 8(3). https://doi.org/10.1002/mbo3.646
  13. Zoqratt, M. Z. et al. 2020. The Microbiota of Malaysian Fermented Fish Sauce. bioRxiv , 2020.03.10.986513. https://doi.org/10.1101/2020.03.10.986513

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