Saving the Bryophytes of Our Forest

Harit Amit Patel

Lai Jin Yan Aimie

Santa Maria Priscilla Nicole

Victoria Junior College

Little Green Dot Student Research Grant

PROJECT REPORT

submitted to

Nature Society (Singapore)

Junior College Category

2012 2845 words

i

Contents:

1. Introduction……………………………………………………………..1

2. Materials and Method………………………………………………….2

2.1 Collection and Identification of Subjects ………………..……....2

2.2 Site Study………………………………………….…..………..….2

2.3 Simulation of Growth…………………….……………………......2

2.4 Preparation of Clean Bryophyte Samples……………………….3

2.5 Water Retention Test.……………………………………………..3

2.6 Preparation of Bryophytes as Test Samples for Anti Microbial

Assay…………………………………………………………....…...3

2.7 Preparation of Microbial Suspension for Anti Microbial

Assay……………………………………………………………..….3

2.8 Preparation of Luria-Bertani (LB) Agar Plates for Bacteria

Culture………………………………………………………..….....4

2.9 Preparation of Potato Dextrose Agar (PDA) Plates for Fungal

Culture………………………………………………………...…....4

2.10 Anti-Microbial Assay……………………………………...……….4

3. Results and Discussion………………………………………………......5

3.1 Identification of Species…………………………………………….....5

3.2 Site Study…………………………………………………………..…...7

3.3 Simulation of Growth…………………………………………….....…7

3.4 Water Retention Test………………………………………….…...….8

3.5 Anti-Microbial Test………………………………………………...….9

4. Conclusion…………………………………………………...…..….….10

5. References ……………………………………...…………………..….11

6. Acknowledgement……………………………………………………..12

7. Appendix

ii

Abstract

Bryophytes are a common sight in Singapore and around the world. However, due to their

relatively diminutive size and a lack of awareness towards them, many properties of

bryophytes may have been overlooked. Three species of bryophytes namely Acrolejeunea

spp., Isopterygium bancanum, Hyophila involuta, have been chosen as the target of our

research project, to investigate into the possible properties such as bryophytes’ water

retention abilities and anti-microbial properties. A site study was done analyzing the pH

and temperature of the environment that bryophytes are commonly found in. followed by

tests on water retention ability and anti-microbial properties. Results have shown that

bryophytes have excellent water retention, and work in a narrow range of pH. The three

bryophyte species did not possess anti-microbial properties. A further extension could be

investigating methods to speed up bryophytes’ growth rate, and the ability of bryophytes’

sap to raise the freezing point of water. (150 words)

1

1 Introduction

Being small and relatively common, bryophytes are often overlooked and understated.

Hence, we have carried out this project to investigate the benefits that bryophyte

purportedly has. In this project, we have chosen to work on the three species of bryophytes

found in the premises of Victoria Junior College and Pasir Ris Town Park.

Bryophytes possess several beneficial properties. They can serve as early indicators of

acidic or basic pollutants in the environment due to the narrow range of pH they can

survive in (Saxena and Harinder, 2004). Some bryophytes such as the Sphangum moss

possess anti-microbial properties and can be used as field medicine. In fact, the Sphangum

moss was used as an effective surgical dressing during the First World War. (Muma, 1979)

Moreover moss is common and easy to harvest and hence provides an economical

alternative to dressings commonly used today. This is especially important in developing

regions where people can ill afford expensive medical care.

Additionally, some bryophytes, such as Hypnum cupressiforme, possess anti-fungal

properties. Thus, bryophytes can be useful in finding effective solutions to combat various

odour and fungal ailments that commonly assail the feet, such as athlete’s foot and other

fungal infections. (Glime, 2007)

Being an urbanized country, Singapore faces the environmental threat of eutrophication due

to the common use of fertilisers in parks. Leaching of nutrients into ponds, rivers or

reservoirs from the banks occurs regularly due to frequent rain. With increased nutrients,

algae bloom on the surface of the reservoir prevents sunlight from reaching the plants at the

bottom of the reservoir. These plants eventually die, and decompose. The decomposition

uses up oxygen that is critical for the survival of aquatic animals in the pond. Animals and

plants continue to die and decompose, setting off a vicious cycle which ultimately disrupts

the entire ecosystem. (Emmett, 2007)

Currently, the common grass Axonopus compressus (Informed Farmers, 2011) is grown

around reservoirs to prevent soil erosion and nutrient leaching. However, the water

retention ability of grass makes it less effective in preventing nutrient leaching. Some

species of bryophytes have been found to have high water retention abilities (Tomas, 2008)

hence, bryophytes may be crucial in protecting the survival of aquatic ecosystems by

slowing down nutrient leaching to prevent eutrophication. This would also help ensure

good water quality in the reservoirs to safeguard public health.

Hence, our group has studied the soil pH and temperature of a habitat with bryophytes and

investigated the water retention abilities and possible anti-microbial activities of three

2

species: (Acrolejeunea sp, Isopterygium bancanum, Hyophila involuta)

This project aims to uncover some of the properties of bryophytes native to Singapore that

could help in solving environmental problems in a bid to raise public awareness of these

seemingly small and insignificant inhabitants of the forest.

Hypotheses

Bryophytes may have

1 a narrow range of temperatures and pH that they require to flourish.

2 excellent water retention abilities.

3 anti-microbial properties.

2 Materials and Methods

2.1 Collection and Identification of Subjects

Bryophytes were collected from three different locations and labelled P, Q & R.

A hand-held microscope (Digital microscope, BW/008-500K) was used to obtain the

overall profiles of the specimens collected while a digital microscope eyepiece

(MicroVision, MV8) was used to obtain the tissue profiles of the leaves at four key

locations: tip, base, centre, and the side for species identification with assistance from Dr

Benito Tan, an expert bryologist.

2.2 Site Study

The temperature and pH reading of the soil was taken at Victoria Junior College, as it was

an area where bryophytes commonly grow. Readings were collected using a data logger

with temperature and pH sensors (Addest Technology) for a period of thirteen days. Data

was collected daily at 0630h and 1600h. The reading at 0630h before sunrise was taken to

be the lowest temperature of the soil for the day because the sun has yet to rise to warm the

soil and it would have undergone a long period of low atmospheric temperature throughout

the night before. Conversely, the reading taken at 1600h was taken to be the highest

temperature of the soil for the day as the soil would have been exposed to intense heating

from sunlight throughout the day until before sunset.

Results were recorded to determine the conditions of the soil and daily fluctuations for

simulation purposes in the following experiment.

2.3 Simulation of growth

Samples of each species were placed on a cotton wool soaked thoroughly in pH buffers of

pH 4.00 (Scharlau S01004), 7.00 (GCE Laboratory Chemicals, E9350) and 9.00 (Scharlau

S01009100 in separate petri dishes sealed with parafilm. This was done with 2 replicates

for each species and pH value.

3

The dishes were left to stand in the Science & Technology Laboratory in Victoria Junior

College for three weeks with quick checks twice every week to ensure that the dishes did

not dry out.

Browning of the bryophytes and any other visible changes were recorded at the end of the

experiment.

2.4 Preparation of Clean Bryophyte Samples

The collected samples of each species were washed under running tap to remove all visible

traces of soil and placed in the ultrasonicator (Elma, Elmasonic E 30 H) for 15 min for

thorough cleaning. The cleaned samples were blotted dry with paper towels for use in later

experiments.

2.5 Water retention test

A measured quantity of each cleaned bryophyte species as described in section 2.4 was left

in the oven (Labcon, 2029k) at 80oC for 7 days to ensure complete drying. The dried

samples were weighed using an electronic balance (OHAVS, GT410) to determine the

water content of fresh samples.

The dried samples were then completely immersed into a beaker of water for 2mins before

being removed and weighed to determine the amount of water absorbed by each sample.

The water retention index of each sample was determined as follows:

where R refers to Water retention index; W refers to the mass of dried sample after water

treatment; and D refers to the dry mass of sample.

The above procedures were conducted twice for each species.

2.6 Preparation of Bryophytes as Test Samples for Antimicrobial Assay

10 g of each cleaned bryophyte as described in section 2.4 was ground with 10 ml of

distilled water using the mortar and pestle to obtain a pulp (concentration: 1 g ml-1

). The

pulp was left to stand for 10 minutes and decanted with the liquid to be tested for anti-

microbial activity.

2.7 Preparation of Microbial Suspension for Anti-Microbial Assay

Three species of bacteria: Bacillus subtillis (Carolina), Eschericia coli(Carolina),

Micrococcus luteus (Carolina), and a species of fungus: Saccharomyces cerevisiae

(Carolina) were used.

4

One loop of microbe was obtained from each stock culture on agar plates and suspended in

1 ml of nutrient broth.

2.8 Preparation of Luria-Bertani (LB) Agar Plates for Bacterial Culture

37 g of LB agar powder (SIGMA, L2897-250G) was added to 1 l of distilled water. This

mixture was autoclaved (TOMY, High-Pressure Steam Sterilizer ES-215) at 121oC and 100

kPa for 15 min. The autoclaved mixture was poured onto the petri dishes and left to cool in

a biosafety level 1 cabinet.

2.9 Preparation of Potato Dextrose Agar (PDA) Plates for Fungal Culture

A potato (160.02g) was sliced and boiled in 500ml of water for 20 minutes. During the

boiling process, 10g of dextrose was added.

The mixture was then filtered through a coffee filter before 10g of agarose powder was

added.

The mixture was placed into the autoclave machine (TOMY, High-Pressure Steam

Sterilizer ES-215) for 30 minutes. After removal, the PDA was poured into petri dishes and

left to cool.

2.10 Anti-Microbial Assay

50 µl of bacterial suspension was spread around evenly on a plate of LB agar separately for

each species, while 50 µl of fungal suspension was spread around evenly on a plate of

PDA. Preparation of the four species of microbial suspensions are described in section 2.7.

20µl of the test sample was added to the centre of the agar plate after spreading the

microbial suspension. The plates were left to incubate (NÜVE, EN055) at 37 oC for four

days. The diameters of zones of clearing at the centre of each plate, if any, were measured

and recorded accordingly.

The 5 test samples used:

bryophyte pulp 1) P, 2) Q and 3) R as described in section 2.6,

4) ampicillin (1 %) for bacterial cultures, or Ziram (fungicide) for fungal cultures as

positive controls and

5) distilled water as negative control.

The procedures described above were carried out again to produce replicates for each

species of microbe and test sample.

This experiment was done in a Biosafety Level 1 cabinet.

5

3 Results and Discussion

3.1 Identification of species

Sample P:

Figure 1 & 2 Sample P (tip)

Figure 3 Sample P (whole leaf)

Sample R:

Figure 4 Sample R (base) Figure 5 Sample R (tip)

Tip

Tip

Base

Centre

Side

Tip

Base

Tip

6

Figure 6 Sample R (centre) Figure 7 Sample R (base)

Sample Q:

Figure 8 Sample Q (whole leaf) Figure 9 Sample Q (base)

The results of the identification process are as follows:

Table 3.1.1 Location of Bryophyte Species Used.

Sample Location (Refer to site photos in Appendix 7.1) Species

P Tree Bark at Victoria Junior College Acrolejeunea spp.

Q Tree Root at Victoria Junior College Isopterygium

bancanum

R Prawning area at Aquarium Iwarna (Pasir Ris

Farmway)

Hyophila involuta

Centre Base

Centre Base

Tip

Side

Base

7

3.2 Site Study

Table 3.2.1The average daily temperature and pH readings at 0630h and 1600h

Parameters Temperature/°C pH

Time 6:30am 4:00pm 4:00pm

Average Reading 26.3 34.1 7.1

From Table 3.2.1, at 0630h, temperature is relatively low with an average of 26.3°C. At

1600h, which is one of the hottest time of the day, is relatively high at 34.1°C. The pH

reading of the site was only taken once a day at 1600h, as pH value of the site is not

expected to vary throughout the day. The pH value of the site is an average of 7.1,

indicating that the environment is neutral in pH.

3.3 Simulation of growth

Table 3.3.1 Growth of Samples under different pH conditions

Sample pH 4 pH 7 pH 9 Deduction

P Turned

yellow

brown

Turned

yellow

brown

with large

patches

of green

Turned

yellow

brown

Yellow brown indicates necrotic tissue. P

can only survive in neutral pH

environment

Q Turned

dark

brown

with

small

patches

of green

Turned

dark

brown

with large

patches

of green

Turned

dark

brown

Dark brown indicates necrotic tissue. Q

can survive in acidic and neutral pH

environments

R Turned

dark

brown

Turned

dark

brown

Turned

dark

brown

Dark brown indicates necrotic tissue. R

did not survive in all three conditions.

Growth inhibitor of R could be present in

all three media.

8

Referring to Table 3.3.1 above, Sample P grows in a relatively neutral environment and is

susceptible to changes in pH. It is only able to grow in a narrow range of pH under neutral

conditions. Hence it could be grown near areas where control of pH is necessary to serve as

early indicators of pH changes as a result of soil quality degradation due to acid rain or

factory waste discharge.

Sample Q grows in a neutral medium and is partially resistant to acidic conditions. Hence,

it can survive in a wider range of pH, from acidic to neutral conditions. This makes Q a

choice species of bryophytes for introduction into areas plagued by acid rain as it is able to

withstand the lower pH of the soil in these areas.

Sample R was unable to survive in all 3 conditions. The buffers used for the pH 4, 7 and 9

media all contained potassium ions (K+). Sample R may be susceptible to K

+ since high

potassium may induce Calcium and Magnesium deficiency in certain plants like

chrysanthemum (Boodley, 1975), we postulate that high potassium concentration could be

inhibiting the growth of R.

3.4 Water Retention test

Table 3.4.1 Water Retention Ability of the Samples

Sample P Sample Q Sample R Axonopus compressus

Water retention

index

15.0 23.4 6.30 2.37

Bryophyte samples P and Q have a significantly higher water retention index of about 7 and

11 times more as compared to Axonopus compressus as seen from Table 3.4.1. Taking

density of water to be 1 g ml-1

, Axonopus compressus can only absorb 2.37 ml of water per

gram. Sample Q is the bryophyte which has the highest water retention index, absorbing

23.4 ml of water per gram, which is almost 11 times the amount that Axonopus compressus

can absorb. Sample R, although having a significantly lower water retention index then

Sample P and Q, can still absorb about 3 times as much water as Axonopus compressus.

This suggests that bryophytes would be a better option to grow on the banks of water

bodies to absorb surface runoff and help prevent eutrophication.

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3.5 Anti-microbial test

Table 3.5.1 Anti-Micobial activity of Samples and Controls

Sample Bacillus subtillis Micrococcus

luteus

Eschericia coli Saccharomyces

cerevisiae

P Lawn growth

observed

Lawn growth

observed

Lawn growth

observed

*No growth

observed

Q Lawn growth

observed

Lawn growth

observed

Lawn growth

observed

*No growth

observed

R Lawn growth

observed

Lawn growth

observed

Lawn growth

observed

*No growth

observed

Ampicillin Zone of clearing

observed

4.53 cm

(diameter)

Zone of clearing

observed

6.96 cm

(diameter)

Zone of

clearing

observed

2.69 cm

(diameter)

Not applicable

Fungicide Not applicable Not applicable Not applicable *No growth

observed

Distilled

Water

Lawn growth

observed

Lawn growth

observed

Lawn growth

observed

*No growth

observed

*There were no results obtained for the experiments done with Saccharomyces cerevisiae as it

could not grow on the Potato Dextrose Agar.

The three samples were tested against the four microbes to check for the presence of

phytoconstituents which these gram (+) and gram (-) bacteria may be susceptible to. The

use of both gram (+) and gram (-) bacteria was to screen for phytoconstituents whose mode

of action may be limited to the inhibition of peptidoglycan cell wall formation, resembling

penicillin, which will affect gram (+) bacteria only. Therefore, two gram (+) bacteria

(Bacillus subtillis and Micrococcus luteus) and a gram (-) bacterium (Eschericia coli) were

used in this experiment.

Saccharomyces cerevisiae, or yeast, is a fungal representative to screen for possible anti-

fungal phytoconstituents in the bryophytes.

There is no zone of clearing observed for Samples P, Q and R as seen in Table 3.5.1, this

suggests that all 3 bryophytes samples do not possess anti-microbial properties. Although a

number of species of bryophytes such as the Sphagnum mosses have been found to possess

antibiotic properties, the three species of bryophytes under study which are commonly

found in Singapore lack such attributes.

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4 Conclusion

Through the various experiments conducted, we have found the three species (Acrolejeunea

spp., Isopterygium bancanum, Hyophila involuta) to have very high water retention abilities,

as demonstrated by the high water index of 15.0 for Acrolejeunea spp., 23.4 for

Isopterygium bancanum , and 6.30 for Hyophila involuta in comparison to 2.37 for grass

(Axonopus compressus).

These three species which are commonly found in Singapore also do not possess any anti-

microbial properties. In addition, we have found that Acrolejeunea spp. is able to survive in

neutral conditions, and Isopterygium bancanum can survive in pH ranging from acidic to

neutral, while Hyophila involuta could not grow in the three buffer solutions - possibly due

to the high potassium content in all the buffer medium used.

Since Isopterygium bancanum has been proven to have the highest water retention index,

and is able to survive in a wider range of pH as compared to the other two species, it is

likely to be effective in curbing eutrophication. Therefore, we propose the reintroduction of

Isopterygium bancanum along river banks, ponds and other water bodies to prevent

eutrophication. One such water body would be the turtle pond at East Coast Park (Figure

7.4 in appendix) which is showing early signs of eutrophication with the green murky water

caused by algal bloom.

Moving forward, we hope to be able to work with more bryophyte species to test their

water retention abilities and continue to search for potential anti-microbial

phytoconstituents. Moreover, as bryophytes are known to have very slow growth rates,

discovering propagation methods to help speed up their growth rates will help

tremendously in ensuring their survival.

Also an accidental discovery in the laboratory revealed that the sap obtained from

Acrolejeunea spp. and Hyophila involuta increased the freezing point of water by up to 7oC.

(Refer to Appendix Figure 7.5) We can hence study and compare this ability of sap from

more species of bryophytes to explore a possible all-natural solution to slow down the

melting of ice caps which would mitigate the problem of rising sea levels.

11

5 References

Boodley, J. W. (1975, April 10). Recognizing Nutrient Deficiency Symptoms. cornell.edu.

Retrieved December 25, 2012, from

http://counties.cce.cornell.edu/schenectady/new/pdf/ag%20fact%20sheets/soil/Recognize%

20Nutrient%20Deficient%20Symptoms.

Emmett, J. (2007, December 18). Eutrophication. In Encyclopedia of Earth. Retrieved

December 25, 2012, from http://www.eoearth.org/article/Eutrophication

Glime, Janice M. 2007. Bryophyte Ecology. Volume 5. Uses. Ebook sponsored by

Michigan Technological University and the International Association of Bryologists.

Rerieved from http://www.bryoecol.mtu.edu/

Informed Farmers (2011, June 23). Broadleaf Carpetgrass - Turf Grass « Informed

Farmers.Welcome to the Informed Farmer « Informed Farmers. Retrieved December 25,

2012, from http://informedfarmers.com/broadleaf-carpetgrass/

Muma, R. (1979). Discovering The Mosses. World of Mosses Website.

Retrieved December 27, 2012, from http://worldofmosses.com/dtm/dtm03.html

Tomas, H. and Hodgetts, N. (2008). Mosses, liverworts, & hornworts. In IUCN. Retrieved

December 25, 2012, from http://data.iucn.org/dbtw-wpd/edocs/2000-074.pdf

12

6. Acknowledgements

To thank Nature Society (Singapore), WWF and IKEA for the sponsorship of the

project and the opportunity to work on a Science Project related to conservation.

Thank the Science & Technology Centre at Victoria Junior College for providing the

research facilities and technical support from Mdm Aslinda and Ms Celina. Thank

Dr Benito Tan for his advice on the research and his assistance with the

identification of the species of bryophytes used in the project.

1

7 Appendix 7.1: Site Photos

Figure 7.1.1: Picture of Tree where Samples P and Q were taken

Figure 7.1.2: Picture of Sample P site: tree bark at Victoria Junior College

Figure 7.1.3: Picture of Sample Q site: tree root in Victoria Junior College

Figure 7.1.4: Picture of Sample R site: Prawning area at Aquarium Iwarna (Pasir Ris Farmway)

7.2 Images of Simulation of Growth experiment Figure 7.2.1: Sample P

A

B

Figure 7.2.1: Figures of experiment for Acrolejeunea sp

A: pH 4, pH 7, pH 9 B: Contrast between pH 4 and pH 7 C: Contrast between pH 7 and pH 9

C

Figure 7.2.2: Sample Q

A

B

Figure 7.2.2: Figures of experiment for Isopterygium bancanum

A: pH 4, pH 7, pH 9 B: Contrast between pH 4 and pH 7 C: Contrast between pH 7 and pH 9.

C

Figure 7.2.3: Sample R

A

B

Figure 7.2.3: Figures of experiment for Hyophila involuta

A: pH 4, pH 7, pH 9 B: Contrast between pH 4 and pH 7 C: Contrast between pH 7 and pH 9

C

7.3 Images of Anti-Microbial Experiment

Figure 7.3.1: Bacillus Subtillis

Figure 7.3.1: Figures of anti-microbial experiment for Bacillus Subtillis

A: Bacillus Subtillis against Sample P, Sample Q and Sample R

B: Bacillus Subtillis against Distilled Water and Ampicillin

Figure 7.3.2: Micrococcus luteus

A

B

A

Figure 7.3.2: Figures of anti-microbial experiment for Micrococcus luteus

A: Micrococcus luteus against Sample P, Sample Q and Sample R

B: Micrococcus luteus against Distilled Water and Ampicillin

Figure 7.3.3: Saccharomyces Cerevisiae

B

A

Figure 7.3.3: Figures of anti-microbial experiment for Saccharomyces Cerevisiae

A: Saccharomyces Cerevisiae against Sample P, Sample Q and Sample R

B: Saccharomyces Cerevisiae against Distilled Water

C: Saccharomyces Cerevisiae against Fungicide

C

B

Figure 7.3.4: E.Coli

Figure 7.3.4: Figures of anti-microbial experiment for E.Coli

A: E.Coli against Sample P, Sample Q and Sample R

B: E.Coli against Distilled Water and Ampicillin

B

A

Figure 7.3.5: Bacteria Pigmentation

Figure 7.3.5: Bacteria Pigmentation

A: Focus on Bacteria Pigmentation on Sample R with E.Coli

B: Focus on gradual Bacteria Pigmentation on Sample Q and R with E.Coli

A

B

Figure 7.4: Turtle Pond at East Coast Park (Proposed site of re-introduction)

B

A

Figure 7.5: Freezing point of Water

A: Samples P, Q and R mixed with distilled water, Sample Q is not frozen

B: Sample P frozen at 4oC

C: Sample Q at 4oC

D: Sample R frozen at 7oC

C

D