Practical 4 : Microalgae Growth Measurement Methods

Introduction

Density of microalgae is widely tested all around the world as it is important as an indicator for a lake or sea to be determine whether the density is too much ( nutrient overload) or too less ( lack of nutrient ) which will lead to the healthiness of the sea. There are several method of calculating the density, for example Dry Weight method ( used to determine the density by weight ), Dilution method ( by using the haemocytometer to count the microalgae) and the most expensive method which is spectrophotometer ( by detecting the light reflection by chlorophyll a ).

Objectives

To determine the density of the stock culture

To determine whether which method has the highest accuracy

Materials and methods

A) Dry Weight method

Picture 1: Amphora sp

Picture 2: Weighing the filter paper (initial weight)

Picture 3: Filtered an exact volume on pretared glass microfibre filters by using a bilchner setup connected to a vacuum pump (Control filters : Seawater)

Picture 4 : The filtered paper are washed with ammonium formate (0.5M) to remove salts. 

Picture 5: The filtered paper are sealed in the aluminium foil and dried at the oven at 100 degree Celsius for 4 hours to volatilize the ammonium formate

*Repeat the steps from picture 2 till picture 5 by using the Amphora sp 

*After 4 hours, the filtered paper are weighted.

B)Chlorophyll Analysis method

Picture 6: 50ml sample are filtered through the pretared glass microfibre filters. 

       Add 3 to 5 drops of MgCO3 to the sample as it is being filtered.

Picture 7: Edges of filter which are not coated with residue being trimmed away. 

Picture 8: The filter with 5ml acetone are grinded for 1 minute. After that, 5ml more of acetone are being added and grinded for another 30 seconds. 

Picture 9: Extract are done and refrigerated in the dark for an hour.

Picture 10: After an hour, the sample was centrifuged at 3000rpm for 10 minutes.

Picture 11: The absorbance of the sample extract are measured by Spectrophotometer at 750nm, 664nm, 647nm and 630nm.

Picture 12: Glass Cuvettes.

Picture 13: Delicate Task Wipers

Picture 14: The extract are inserted by using a droplet at the volume of 5ml into the Cuvette.

Picture 15: The Cuvettes are wiped before inserted into designated place

Picture 16: The Cuvettes are inserted into the spectrophotometer.

Picture 17: The program are set and ready to go.

Picture 18: Results are obtained.

C) Dilution method

Picture 19: 6 test tube are prepared

Picture 20: Stock culture are inserted in 5 test tube which are in different volume, 1 ml, 2 ml, 3 ml, 4 ml, and 5 ml.

*5ml stock culture does not need to be filled with seawater*

Another 1 test tube are filled in with 5ml of seawater as constant, the rest are filled with seawater until 5 ml of the total volume

Picture 21: Test tube are labeled

Picture 22: Haemocytometer are used to count the numbers of Amphora under microscope.

Results

1)Estimation of Dry weight

Weight(g)	Filter with seawater	Filtre with microalgae
Before	0.1	0.1
After	0.10	0.11

2) Chlorophyll analysis

Wavelength(wm)	630	647	664	750
Absorbance	0.0432	0.0567	0.0431	0.0204

3)Optical density analysis

%	Cell/ml	OD
0%	0	0
20%	9 000	0.0732
40%	150 000	0.1622
60%	260 000	0.2689
80%	360 000	0.2919
100%	1 000 000	0.3775

Discussion
Due to the natural characteristic of  Amphora sp. to be a benthic microalgae, it is common for it to easily clump together and descend to the bottom of a container which may affect a reading when the sample is not continuously mixed or stirred. In each method, the Amphora sp. culture is constantly stirred to achieve a more accurate result.

Field Trip: Aquascape Paradise

Aquascape is the arts of arranging aquatic plants as well as rock, stones, driftwood and so on. Other people call it as underwater gardening. Nomally, an aquarium would be decorate with fish plants and all sorts of creative idea that can be come out with. With our field trip to Aquascape Paradise we learned various ways to make underwater art using freshwater macrophytes that are truly outstanding.

Commonly used species are Blyxa sp., Myriophyllum sp., Hydrilla verticillata, Alternanthera sp., Anubias nana, Bacopa carolinianna, Hydrocotyle verticillata, H. leucocephala, Hemianthus sp.,  Java fern, Java moss and others which includes about 114 local species and imported species to create underwater concept. 

Materials that can be used

Driftwood

Rock imported from japan which have extra nutrient

Designs of the aquascape

There are different design outside there mostly based on japan and dutch

Paludariums: 

An aquarium that combines water and land inside the same environment.

Jungle style:

Natural, untrimmed look that provide wild appearance

Iwagumi style:

Main layout materials are stones and covered with mosses

Dutch style

By having multiple plants with different colour, and the tank have more than 80% covering the aquarium floors

Nature style:

Mountain like landscape

Types of aquatic plants

Mosses tied on the driftwood that resembles a tree

Terrarium

Example of how the shop grow their aquatic plants

Water are sprinkled from above to keep the soil and plant moisture

Special thanks to the management of Aquatic Paradise for the warm welcome and knowledge on aquascaping.

Practical 5: Propagation of Seaweed in Culture Media

Introduction
Seaweed are know to reproduce through fragmentation where if some parts of its thallus are torn or broken apart, they are able to regenerate and grow a new thallus. This is process is also carried out symbiotic with bacteria that supplies nutrients such as nitrogen to the seaweed. This study is to observe the species of bacteria that assist in fragmentation.

Objective
To determine which bacteria is best for the culturing the seaweed

To learn ways to propagate the seaweed

Material and Methods

Picture 1:Cut 1 cm of the seaweed

Picture 2: Rinsed the seaweed by using filtered sea water and shaken vigorously to sterilized the seaweed

Picture 3: Record the wet weight of seaweed

Picture 4: Inserted the culture media Von Stosch's Enrichment (VSE) and Provasoli Enrichment Seawater (PES)

Picture 5: Inserted the bacteria ( 4 types of bacteria)

Picture 6:Insertion of bacteria to algae sample.

Picture 7: Sealed the side of the container using parafilm

After that, incubate the seaweed for a photoperiod of 12 hr light and 12 hr dark with aeration

Results

Picture 8: First day of propagation

Picture 9: One week after the propagation

Picture 10: Few week after the propagation the buds begin to emerge

Discussion
The first week does not shows much growth or we can say probably no growth as there no new shoots growing out, however, the result can be seen after a few weeks as the shoot grow as long as 1 cm as shown in BAC3 which shows positive budding as the algae is associated with BAC3.