Application

• STAL1023


  Introduction to Marine Flora
• Outlines


  • Sampling & Identification
  • Algal isolation & culture techniques
  • Preservation of algal samples
  • Quantitative determinations of algal density & growth
  • Growth rate & generation time determination
• Sampling


  The Collecting, Cleaning, and Mounting of Diatoms
• Preservation of algal samples

  Culture/ Identification/ Collection<|>Live samples<|>Storage periods requires addition of chemicals that reduce: Bacterial activity, Autolysis of algal cells
• Preservatives


  • Formalin
  • Lugol's solution
  • Gluteraldehyde
• Lugol's Iodine Solution


  1% final concentration (1 part per 100)<|>Bacteriostatic, but does not preserve structure<|>Iodine will bind to starch and form blue black<|>It can be used as a cell stain, making the nuclei more visible

  
• Glutaraldehyde


  3%, neutralized pH 7<|>70% : for use in ultrastructure studies<|>0.5% : for wall-less flagellates to avoid shrivel & disintegration<|>Wash the glutaraldehyde off with micropore filter apparatus & buffer wash before count/identification<|>If using settling chamber, cover chamber with glass to avoid exposure to glutaraldehyde fumes

  
• Acid-cleaning procedure (Renberg 1990)

  1. Pipette 1ml of sample into 1.5 a microcentrifuge tube, discard excess water

  2. Add 1 ml 30% H202 and put on heat (85 Celcius) for 1-3 hours. This step is to oxidize the outer mucilage layer of frustules

  3. Transfer sample into a bigger tube and add distilled water

  4. Centrifuge to discard liquid

  5. Add 10% HCl to dissolve protoplasm and other organeles, dissolve any calcium and iron compounds

  6. Let the diatom to settle to the bottom of tube, remove HCl by pippetting

  7. Rinse with distilled water 3x

  8. Store sample in 70% ethanol

  
• The most effective method for assaying many diatoms from natural collections requires cleaning
• Distinctive frustule markings are obscured by cell contents
• The most effective methods for making permanent mounts of diatoms require strong acids
• Basics of Identification


  Make a list of genera that you can confidently identify on the basis of: Cell / colony shape, Presence of stalks<|>Consult references as needed
• Algal Isolation


  1. Streaking

  2. Spraying plates

  3. Single cell isolation

  4. Serial Dilution
• Culture Establishment


  Essential for conducting competition studies, bioassays, assessment of zooplankton food preferences, determination of algal life histories<|>Necessary for molecular systematic work<|>Unialgal : contain only one alga<|>Non-axenic : contain bacteria, fungi, protozoa<|>Axenic : only alga<|>Antibiotics to discourage contaminating bacteria<|>Germanium dioxide will inhibit growth of diatoms
• Single cell isolation from aquatic samples

  1. Pure starter culture establishment

  2. Mass cultivation

  3. Cell harvest and Extraction
• Monospecific Microalgae Cultivation


  The most important parameters regulating algal growth are nutrient quantity and quality, light, pH, turbulence, salinity and temperature<|>Limiting factor: Light, N, P, others
• Measuring growth


  Estimating biomass: understanding how matter and energy move between populations<|>It reflects the amount of sunlight, water and minerals a primary producer is able to capture and turn into mass<|>Gives an idea of the state of the ecosystem
• Weighing biomass: Destructive


  1. Wet weight: filter a volume of culture onto a pre-weighed filter pad. Subtract the mass of the pad from the algae and the pad and that is your wet weight

  2. Dry weight: More accurate, pad is often washed with fresh water to remove salts then oven dried at 100C until all the water is driven from inside of the cells

  3. Ash-free dry weight: to know exactly how much organic matter is in the biomass; how much non-biological cellular material the cell has in them
• Chlorophyll-a


  A rapid method for determining phytoplankton density in a sample; involves the extraction and measurement of chlorophyll concentrations<|>The amount of pigments as chlorophyll a, b, c and phaeophytin is considered as a measure of phytoplankton biomass<|>After the collection of the sample, it is filtered through a Millipore (Pore size 0.45 µm) or glass fiber (1 µm mesh) filter, and is pumped to dryness<|>The filter containing the sample is placed in 90% acetone in a plastic vials covered by aluminium foil and shaken vigorously and gently ground with a homogeniser to ensure dissolving of the filter (Millipore) before storage in the refrigerator for 20-24 hr<|>The solution is centrifuged for about 20 minutes at 5000 rpm and the supernatant solution is considered for the determination of optical density<|>Absorbance values with selected wavelengths namely 664 nm, 647 nm, and 630 nm
• Counting Algae in Mixed Assemblages: Sedgwick Rafter Chamber (SRC)


  To determine the number of plankton in a 1 ml sample of water
• Biovolume


  Algal counts can be misleading because the cells vary in size from very tiny (<2 µm diameter) to nearly macroscopic (>1 mm diameter). In addition, some cells are too tiny or indistinct to count accurately, so the algae are counted as colonies<|>Algal biovolume is often used to adjust for variations in size by estimating the total volume occupied by each type of algae<|>In comparison with automated methods, the use of microscopical measurements allows high taxonomic resolution, up to the species level, and has fewer sources of error
• Counting Unialgal Samples: Hemocytometer


  When a cover-slip is placed on the slide, the volume of liquid is fixed, and you can find the number of cells per area<|>By multiplying to the volume of the sample, you can estimate the population of cells
• Growth phases


  The condition of the innoculum has a strong bearing on the duration of the lag phase<|>An innoculum taken from a healthy exponentially growing culture is unlikely to have any lag phase when transferred to fresh medium under similar growth conditions of light, temperature and salinity<|>In general, the length of the lag phase will be proportional to the length of time the innoculum has been in phases 3-5<|>A lag phase may also occur if the innoculum is transferred from one set of growth conditions to another
• Exponential Phase


  The growth rate of a microalgal population is a measure of the increase in biomass over time and it is determined from the exponential phase<|>Once the growth phase has been plotted (time on x-axis and biomass on logarthmic y-axis) careful determination of the exponential (straightline) phase of growth is needed<|>Two points, N1 and N2, at the extremes of this linear phase are taken and substituted into the equation<|>For comparing the results of bioassay experiment; or growth of various algae under the same or varying conditions<|>Cell density data are usually obtained with a hemocytometer or electronic particle counter<|>Growth rates obtained by sampling cultures at intervals. E.g once per day<|>Plot of number of cells against time (in days)
• Feedstock for biofuel production

  The lipid content of Euglena (mainly wax esters) is seen as a promising feedstock for production of biodiesel and jet fuel<|>Under the aegis of Itochu, a start-up company called Euglena Co., Ltd. has completed a refinery plant in Yokohama in 2018, with a production capacity of 125 kiloliters of bio jet fuel and biodiesel per year
• Human Consumption


  The taste of powdered euglena is described as dried sardine flakes, and contains minerals, vitamins and docosahexaenoic, an omega-3 acid. The powder is used as ingredient in other foods to make it more healthy
• Applications


  • Bioprospecting
  • Forensics
  • Paleontology
  • Diatomaceous earth/ diatomite
  • Bioremediation
• Applications


  • Carotenoid: Antioxidant
  • Nano O2: Wastewater Treatment
  • Silica: Filter
  • Silica: Nutritional Application
  • Fatty Acids: Feedstock
  • Lipid: Biodiesel
  • Silica: Biomaterial
• Biological Monitoring


  Different species within a genus may occur in different habitats (marine, freshwater, wastewater ponds etc)<|>Biological monitoring involving identification to genus level and based on community assemblages rather than individual taxa<|>If a stream sample containing ten or more algal taxa that are listed in the "Good quality streams" = healthy algal community<|>If future monitoring in the same stream shows that the algae community has become dominated by taxa more typical of wastewater treatment ponds, should identify the source of nutrients and algae to assess the issue<|>Detection of bloom of cyanobacteria that are potentially toxic<|>As well as the composition of algal communites, the quantity of algae at a site is important
• Innoculum

  
  
• A suspension of microbial cells (bacteria, yeast, etc.) used to start a culture or infection.
• Lag Phase


  
  
• A period of slow or no growth when the innoculum is transferred to a new environment.