Phytobenthos
Source: HIMOM
Background
Phytobenthos refers to the algae that may be found on/in the seafloor sediments. Sediments are sampled and the algal pigments are quantified by spectrophotometer and by HPLC. The sampling strategy is a key issue in these studies due to non-uniform vertical and horizontal distribution of the phytobenthos. In muddy sediments, phytobenthos is found mostly in the top 1.5 cm of sediment whereas in sandy sediments it is possible to find phytobenthos as deep as 15 cm depth (Cartaxana et al., 2006). Phytobenthos distribution may be very patchy and the sampling strategy should take this into account.
No extraction procedure exists that will provide 100% extraction efficiency. Efficiency varies with species and acts differently for the various pigments. The 90% acetone as recommended in this protocol generally provides ca. 90% efficiency (Wiltshire et al. 2000). Moreover, acetone extraction is especially efficient for polar pigments, like chlorophyll a, and thus can be used for the analytical methods described below. Unfortunately, some extraction-resistant species such as cyanobacteria and Zostera exist on the tidal flats, which require mechanical (e.g. grinding) and aggressive chemical (e.g. toxic dimethylformamide and methanol) means to extract these pigments. However, due to other problems associated with these extraction methods, they will not be described here.
Required
- Freezer - centrifuge
- Freeze-Dryer - ultrasound bath
- balance
- 90% buffered acetone
- ultrasound bath
Sampling:
A small petri dish is used to take surface sediment samples at intertidal sites. Ideally, the petri dish should have 20 ml volume and a height of 2 cm. The Petri dish is placed on the sediment and collected by means of a plastic card that is slid under the plate to break the contact with underlying sediment.
This method works very well with mud, however, for sampling sand, may want to sample a deeper depth, based on previous (preliminary) experience regarding vertical distribution of the phytobenthos. If sampling in a subtidal area, sediments may be taken by divers, by Craib corer or by box corer (sediment surface must NOT be disturbed) and then subsampled using the Petri dish method.
Lab work:
Extraction should always be carried out under dim light, keeping the samples and solvent cold/on ice.
1. Sediment samples should be frozen as soon as possible and then freeze-dried
2. Weigh out an amount (about 15g) of freeze-dried sediment and transfer to 50ml centrifuge tube
3. Add about 10% (w/w) quartz sand (MERCK- particle size 10-30µm) to the tube
4. Add 15ml acetone (10% saturated sodium carbonate : 90% pure acetone) to the tube.
5. Place the tubes in an ultrasound bath containing -4ºC water for 90 minutes.
6. Place the sealed tube with sample in a freezer (-20ºC) to extract for 24 hours (vortex after that).
7. At end of 24 h, vortex the tubes and centrifuge 10 min at 3000 rpm.
8. Take the extract for pigment analysis, using HPLC or spectrophotometer.
References
Bowles, N. D., Paerl, H. W. & Tucker, J. 1985. Effective solvents and extraction periods employed in phytoplankton carotenoid and chlorophyll determinations. Can. J. Fish. Aquat. Sci. 42: 1127-31
Cartaxana, P., Mendes, C.R., van Leeuwe, M.A & Brotas, V., 2006. Comparative study on microphytobenthic pigments of muddy and sandy intertidal sediments of the Tagus estuary. Est. Coast. Shelf Sci. 66: 225-230.
Wiltshire, K.H., M. Boersma, A. Molle & H. Buhtz, 2000. Extraction of pigments and fatty acids from the green alga Scenedesmus obliquus (Chlorophyceae). Aquatic Ecol. 34: 119-126.
Wright, S. W., S. W. Jeffrey & R. F. C. Mantoura, 1997. Evaluation of methods and solvents for pigment extraction. In: Jeffrey, S. W., Mantoura, R. F. C., and Wright, S. W. [Eds], Phytoplankton pigments in oceanography: guidelines to modern methods. UNESCO, Paris, pp. 261-82.
Background
Phytobenthos refers to the algae that may be found on/in the seafloor sediments. Sediments are sampled and the algal pigments are quantified by spectrophotometer and by HPLC. The sampling strategy is a key issue in these studies due to non-uniform vertical and horizontal distribution of the phytobenthos. In muddy sediments, phytobenthos is found mostly in the top 1.5 cm of sediment whereas in sandy sediments it is possible to find phytobenthos as deep as 15 cm depth (Cartaxana et al., 2006). Phytobenthos distribution may be very patchy and the sampling strategy should take this into account.
No extraction procedure exists that will provide 100% extraction efficiency. Efficiency varies with species and acts differently for the various pigments. The 90% acetone as recommended in this protocol generally provides ca. 90% efficiency (Wiltshire et al. 2000). Moreover, acetone extraction is especially efficient for polar pigments, like chlorophyll a, and thus can be used for the analytical methods described below. Unfortunately, some extraction-resistant species such as cyanobacteria and Zostera exist on the tidal flats, which require mechanical (e.g. grinding) and aggressive chemical (e.g. toxic dimethylformamide and methanol) means to extract these pigments. However, due to other problems associated with these extraction methods, they will not be described here.
Required
- Freezer - centrifuge
- Freeze-Dryer - ultrasound bath
- balance
- 90% buffered acetone
- ultrasound bath
Sampling:
A small petri dish is used to take surface sediment samples at intertidal sites. Ideally, the petri dish should have 20 ml volume and a height of 2 cm. The Petri dish is placed on the sediment and collected by means of a plastic card that is slid under the plate to break the contact with underlying sediment.
This method works very well with mud, however, for sampling sand, may want to sample a deeper depth, based on previous (preliminary) experience regarding vertical distribution of the phytobenthos. If sampling in a subtidal area, sediments may be taken by divers, by Craib corer or by box corer (sediment surface must NOT be disturbed) and then subsampled using the Petri dish method.
Lab work:
Extraction should always be carried out under dim light, keeping the samples and solvent cold/on ice.
1. Sediment samples should be frozen as soon as possible and then freeze-dried
2. Weigh out an amount (about 15g) of freeze-dried sediment and transfer to 50ml centrifuge tube
3. Add about 10% (w/w) quartz sand (MERCK- particle size 10-30µm) to the tube
4. Add 15ml acetone (10% saturated sodium carbonate : 90% pure acetone) to the tube.
5. Place the tubes in an ultrasound bath containing -4ºC water for 90 minutes.
6. Place the sealed tube with sample in a freezer (-20ºC) to extract for 24 hours (vortex after that).
7. At end of 24 h, vortex the tubes and centrifuge 10 min at 3000 rpm.
8. Take the extract for pigment analysis, using HPLC or spectrophotometer.
References
Bowles, N. D., Paerl, H. W. & Tucker, J. 1985. Effective solvents and extraction periods employed in phytoplankton carotenoid and chlorophyll determinations. Can. J. Fish. Aquat. Sci. 42: 1127-31
Cartaxana, P., Mendes, C.R., van Leeuwe, M.A & Brotas, V., 2006. Comparative study on microphytobenthic pigments of muddy and sandy intertidal sediments of the Tagus estuary. Est. Coast. Shelf Sci. 66: 225-230.
Wiltshire, K.H., M. Boersma, A. Molle & H. Buhtz, 2000. Extraction of pigments and fatty acids from the green alga Scenedesmus obliquus (Chlorophyceae). Aquatic Ecol. 34: 119-126.
Wright, S. W., S. W. Jeffrey & R. F. C. Mantoura, 1997. Evaluation of methods and solvents for pigment extraction. In: Jeffrey, S. W., Mantoura, R. F. C., and Wright, S. W. [Eds], Phytoplankton pigments in oceanography: guidelines to modern methods. UNESCO, Paris, pp. 261-82.
