Calcium Uptake of Filamentolus Microalgae in Different Levels of Light Intensity

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The Osaka Gas Foundation of International Cultural Exchange (OGFICE)

Research Grant FY 2013/2014

Final Report

Calcitnn Uptake of

Fila

Jnentolls Microalgae

in

Different Levels of Light Intensity

1. Hefnl Effendi

Center for Environmental Research, Bogor Agricultural University

2. Majariana Krisanti

Water Resource Management Departemen, Faculty of Fishery,

Bogor Agricultural University

3. Nlken T .M. Pratiwl

4. Inna Puspa Ayu

Submitted to:

Center for Environmental Research Bogor Agricultural University

for

The Osaka Gas FoundaHon of InternaHonal Cultural Exchange (OGFICE) October 2014

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General Information

Title

Researchers: 1. Name

Institution

Address of institution

2. Name

Institution

3. Name

Institution

4. Name

Institution

: Calcium uptake of filamentous microalgae in different levels of light intensity

: Dr. Hefni Effendi, M.Phil.

: Center for Environmental Research, Bogar Agricultural University

: Gedung PPLH

Lt.

2-4, JI. Lingkar Akademik, Kampus IPB Dramaga, Bogor 16680. E-mail: [email protected] Phone. 0251-8621262; Fax. 0251-8622134

: Dr. Majariana Krisanti, S.PL M.Si.

: Water Resource Management Depariemen, Faculty of Fishery, Bogor Agricultural University

: Gedung Fakultas Perikanan dan IImu Kelautan Level 3

Wing 7, JI. Agatis Kampus IPB Dramaga, Bogar -Indonesia Phone. 0251-8624360; Fax. 0251-8622932

: Dr. Ir. Niken T.M. Pratiwi, M.Si.

Wing 7, JI. Agatis Kampus IPB Dramaga, Bogar - Indonesia Phone. 0251-8624360; Fax. 0251-8622932

: Inna Puspa Ayu, S.Pi, M.Si.

Wing 7, JI. Agatis Kampus IPB Dramaga, Bogar -Indonesia Phone. 0251-8624360; Fax. 0251-8622932

Center for Environmental Research,

Bogor Agricultural University,

Director,

Bogor, October 2014

Principal Investigator,

Dr. Hefni Effendi, M.Phil. Dr. Hefni Effendi. M.Phil.

•

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Executive Summary

Nowadays, supply of high quality live feed in hatchery and nursery is a necessary need. In molting phase Crustacean needs more calcium in their live feed to reach high survival rate. Enrichment in live feed is the rapid way to absorb mineral for aquatic organism. The possibility of live feed enriched with those mineral is microalgae. Filamentous microalgae could grow in a condition when calcium concentration is in maximum level. This phenomenon is match with the need of enrich live feed for crustacean in molting phase. The objective of this project is to study calcium uptake by

filamentous microalgae in different level of light intensity in laboratory scale.

Filamentous microalgae are selected because the presence in waters usually becomes blooming and useless, short period of growth phase, and fast to harvest. Supply of high calcium microalgae as live feed for commercial aquatic biota could be

achieved, especially in hatchery and nursery activity. The selected filamentous algae is Spirogyra sp. Cultivation of selected genus filamentous microalgae is succeeded under

laboratory condition.

This study consist of three experiments in laboratory, under controlled environment. The first experiment is treatment of selected filamentous microalgae

(Spirogyra sp.) cultivation in various kind of calcium (CaCI2, CaSO., and CaC03). The second experiment is treatment of Spirogyra sp. cultivation with calcium (CaCh) in various kind of concentration and different levels of light intensity, and the last eperiment is treatment of Spirogyra sp. cultivation in absorbing calcium (CaCh) with different irradiation time.

The results of present study showed that CaCI2 is the most effective type of calcium that absorbed by Spirogyra sp. The best CaCI2 concentration added in Spirogyra cultiva tion was

0.5

mL/L medium at the optimum light intensity (500-750 II cd). The concentration of Ca Spirogyra sp. increased 23.4% and 21,8% in treatment 12

hours/day and 24 hours/day, respectively, but final weights in treatment 24 hours/day greater than 12 hours/day. Suspected that Ca is an element that is not needed in large amounts by Spirogyra sp. in their growth process.

Spirogyra sp. which is contain high calcium could apply as live feed in hatchery and nursery, especially for molting phase and increase of survival rate of aquatic biota.

Value added for filamentous microalgae, utilization of filamentous microalgae as valuable product not as a weed. Utilization of filamentous microalgae as natural resources is a chain of renewable energy exploration.

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TABLE OF CONTENT

General Information ...

i

Executive Summary ...

ii

Table of content ...

iii

List of table ... iv

List of figure ... iv

I. Introduction ... 1

1.1. Background ... 1

1.2. Objectives ... 1

II. Literature Review ... 2

11.1. Filamentous Algae ... 2

11.2.

Calcium ...

3

11.3.

Light Intensity ...

3

III . Reseach Method ... 4

111.1. Sampling and isolation of microalgae ... 4

111.2.

Research in Laboratory ...

5

111.3.

Data Analysis ...

8

IV. Results and discussion ... 9

IV. 1. Result of Experiment 1 Treatment of selected filamentous microalgae (Spirogyra sp.) cultivation in various kind of calcium (CaCI2. CaS04. and CaC03) ...

9

IV.2. Result of Experiment 2. Treatment of selected

Spirogyra

sp. cultivation with calcium (CaCI2) in various kind of concentration and different levels of light intensity ... 11

IV.3. Result of Experiment 3. Treatment of selected

Spirogyra

sp. cultivation in absorbing calcium (CaCb) with different irradiation time ... 18

IV.4. Discussion. ... .... ... ... ... ... ... ... ... ... ... ... ... .... ... ...

23

V. Conclusion and recommendation ... 24

V .1. Conclusion ... 24

Acknowledgement ... 25

References ... 25

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LIST OF TABLES

Table 1. Range of pH values during the study ... 14

Table 2. Range of temperature during the study... 15

LIST OF FIGURES

Figure 1. Filamentous algae: Spirogyra sp ... 2

Figure 2. Filamentous algae in pond ... 4

Figure 3. Isolation microalgae in laboratory ... 4

Figure 4. Isolate of microalgae, Spriogyra sp ... 5

Figure 5. Aclimatitation and cultivation of Isolate of microalgae, Spriogyra sp ... ... ... 5

Figure 6. Various kind of calcium treatment on Spirogyra sp. Cultivation ... 6

Figure 7. Various kind of light intensity treatment on Spirogyra sp. Cultivation ... 7

Figure 8. Different irradiation time treatment on Spirogyra sp. 24 hours/day (left) and 12 hours/day (right) ... 8

Figure 9. Calcium concentration in water on Spirogyra sp. cultivation with various kind of calcium ... 9

Figure 10. Calcium concentration in Spirogyra sp. on Spirogyra sp. cultivation with various kind of calcium ... 10

Figure 11. Fresh weight of Spirogyra sp. on Spirogyra sp. cultivation with various kind of calcium ... 10

Figure 12. Calcium concentration in water on Spirogyra sp. Cultivation with various kind of concentration and different levels of light intensity ... 12

=igure 13. Calcium concentration in Spirogyra sp. on Spirogyra sp. cultivation with various kind of concentration and different levels of light intensity ... 13

=tgure 14. Fresh weight of Spirogyra sp. on Spirogyra sp. cultivation with various kind of concentration and different levels of light intensity ... 14

=tgure 15. Value of light intensity during the study ... 15

ｾｧｵｲ･＠ 16. Conditions of cells at the start of treatment (day 0) ... 16

•

[image:6.1496.52.1447.125.1689.2]

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=.gure 17. Conditions of cells at the end of treatment (day 7) ... 17 =igure 18. Calcium concentration in water on Spirogyra sp. cultivation

in absorbing calcium (CaCh) with different irradiation time ... 18 =igure 19. Calcium concentration in Spirogyra sp. on Spirogyra sp.

cultivation in absorbing ca lcium (CaCI2) with different

irradiation time ... 19

=igure 20. Fresh weight of Spirogyra sp. on Spirogyra sp. cultivation in

absorbing calcium (CaCI2) w ith different irradiation time ... 20

ｾＮｧｵｲ･＠

21.

Photo cell Spirogyra sp. light treatment

12

hours/day ...

21

=igure

22.

Photo cell Spirogyra sp. light treatment

24

hours/day ... 22

•

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I.

INTRODUCTION

1.1. Background

Supply of live feed in hatchery and nursery is a necessary need. Live feed which means microalgae is a basic feed in crustacean molting phase. Molting phase is a crucial period for crustacean. In that phase, Crustacean needs more calcium in their feed to reach high survival rate. Enrichment in live feed is the rapid way to absorb mineral for aquatic organism. The possibility of live feed enriched with those mineral is microalgae.

In other side, there are microalgae that could grow well with excess calcium condition. In natural waters, filamentous microalgae could grow in a condition when calcium concentration is in maximum level. This phenomenon is a key to answer the need of enrich live feed for crustacean in molting phase. Especially, filamentous -microalgae is useless aquatic microorganism, just a few of genus that already useful. Utilization of filamentous microalgae as natural resources is a chain of renewable energy exploration.

1.2. Objectives

This research aim to cultivate of filamentous microalgae with different level light in tensity in pure culture, which is related with calcium uptake in order to supply high calcium microalgae as live feed for commercial aquatic biota.

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II. LITERATURE REVIEW

11.1. Filamentous Algae

Filamentous algae are a group of green algae (chlorophyta) with special features thallus such as filament-shaped, branched and unbranched. Filamentous algae commonly found in fresh waters in large numbers. These organisms typically attached, but may detach to become planktonic usually live floated on the surface of the water in len tic ecosystem, or in the lotic ecosystem with relatively small flow. Bellinger and Sigee (2010) explain that in smaller ponds, filamentous Spirogyra sp. and colonial Hydrodictyon frequently form surface blooms or scums

Reproduction of filamentous algae generally by vegetative (fragmentation of filament) and sexsual reproduction by conjugation (Kumar and Singh 1979). Sexual and asexual reproduction such as occurs in Spirogyra sp. (Bellinger and Sigee 2010). Filamentous algae capable of photosynthesis through the utilization of existing nutrients and act as primary producers in the waters. Filamentous algae generally be natural

feed for fish.

Spirogyra sp. is one of filamentous algae, that live in the freshwater habitats. According to Hujaya (2008), Spirogyra sp. have a size cell between 10-1000 micrometers, which are connected to each other from end to end without branching so that it appears as a filament. Cell wall consists of two layers, the outer layer is formed of cellulose while the inner wall is formed from pectin. The size of these filaments can reach several centimeters in length (Figure 1).

Figure 1. Filamentous algae: Spirogyra sp.

Spirogyra sp. in one study were found have a chlorophyll content about 0.53% by weight of the dry sample (Hujaya 2008). This varies depending on the availability of

nutrients, climate and weather conditions on the ecosystem.

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11.2. Calcium

The mineral composition of water is the most important environmental factor that affects the productivity, development, growth, stability, and physiological and biochemical processes of aquatic organism (Martem'yanov & Mavrin 2012).

Calcium plays a pivotal role in the signal transduction of many responses in plant cells (Hepler and Wayne 1985). Jacobshagen et al. (1986) explain that the calcium ion

has been implicated in light-stimulated chloroplast movement responses in Mesotaenium and filamentous algae Mougeotia. Extracellular calcium is necessary for

chloroplast rotation and reduction of intracellular calcium inhibits light-stimulated chloroplast reorientation in the filamentous alga Mougeotia (Wagner and Klein 1978).

11.3. Ught Intensity

Light is an energy source that plays an important role in the primary productivity. Through the primary productivity of this energy will be fixed by autotroph organism through photosynthesis. Based on the physiology of phytoplankton spectrum of light that plays an important role in photosynthesis only ｴｨｾ＠ spectrum with a wavelength of 380-700 nm, known as photosynthetic active radiation (PAR).

Generally, algae showed different responses to changes in light intensity. Pigment chlorophyll absorbs blue and red light, absorb carotene absorbs blue and green light, phycoeritrin absorb green and yellow light absorbing phycocyanin. Optimum light intensity for growth of Chlorophyta is 500-750 foot candella (Ryther 1956).

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III. RESEARCH METHOD

111.1. Sampling and isolaHon of microalgae

1. Sampling of filamentous microalgae

-Sampling of filamentous microalgae from inland water around Bogor, West Java. Filamentous microalgae were collected from natural pond in Cikampak village, Ciampea, Bogor and also from collection pond in Bogor Botanical Garden, West Java, Indonesia.

Physical observation of filamentous microalgae in natural waters and match the characteristic of filamentous microalgae with literature.

&

./

Figure 2. Filamentous algae in pond

2. Cultivation of filamentous microalgae

-Identification of filamentous microalgae which can possibly culture Isolation of selected genus filamentous microalgae

Figure 3. Isolation microalgae in laboratory

[image:11.1492.39.1450.88.2263.2]

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•

_•

r-

I

Figure 4. Isolate of microalgae, Spriogyra sp.

- Microalgae cultivation in laboratory scale with selected media

Figure 5. Aciimatitation and cultivation of Isolate of microalga e, Spriogyra sp.

111.2. Research In Laboratory

Experiment 1. Treatment of selected filamentous mlcroalgae (Spirogyra sp.) culHvaHon

In various kind of calcium (CaC!', CaSO •. and CaCO. )

The study was conducted through experiments on a laboratory scale. There were 4 trea tments: control, CaCI2 treatment, CoSO. treatment, and CaC03 treatment. Three replicates for each treatment.

This study used the glass jars that sterilized by KmnO •. This study used 1 L of sterile water to the jar and added 0.1 mL of fertilizer Gandasil D® in each jar. Added calcium CaCI2 (36,76 g CaCb.2H20 dissolved to 1 L of water. and then used 1 mL for 1 L media), CaSO. (60 g CuSO •. 2H20 dissolved to 1 L of water. and then used 1 mL for 1 L media). and 0,076 g CaC03 for 1 L media, appropriate treatment labels on jars. 1.5 g of Spirogyra sp. added on each jar (Brubaker et

01.

2011; Hmaidan et

01.

2011; Roman et 01. 2011). The jar is placed on a cultivation shelf. equipped with aeration and lights.

During the 7 days retention. pH, temperature, and light intensity were measured daily. Fresh weight of Spirogyra sp.. Co concentration in the water, and the

[image:12.1497.21.1450.40.1213.2]

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concentration of Ca in Spirogyra sp. measured at the beginning and at the end of the study.

Figure 6. Various kind of calcium treatment on Spirogyra sp. cultivation

Experiment 2. Treatment of selected Sp;rogyra sp. cultivation with calcium (CaCb) In various kind of concentration and different levels of light Intensity

Based on the results of previous studies related to the type of calcium is best absorbed by Spirogyra sp .. calcium used in this study is CaCb. CaCI2 were taken from CaC12.2H20 36.76 g dissolved in I L of water.

The study was conducted through experiments on a laboratory scale. Treatment differences were tested. namely the concentration of calcium is added (0 milL. 0.5 mL/L. I mL/L. and 2 mL/L) as well as differences of light intensity (minimum light intensity <500 ft cd. the maximum light intensity> 750

It

cd. as well as the optimum light intensity for growth Chlorophyta 500-750 ft cd (Ryther 1956). Do three replicates for each

reatment.

Once the container jars ready. inserted 2 L of sterile water and added 0.2 mL of ertilizer Gandasil D® on each jar (0.1 mill). Added calcium (0 milL. 0.5 mL/L. 1 mL/L. and 2 mL/L) appropriate treatment label on the jar. Furthermore. as many as 3 g Spirogyra sp. included on each jar (1.5 g / L). a jar placed on a shelf next to the light

intensity of different cultures and aerated.

During the 7 day retention. measured pH. temperature. and light intensity daily. Weight of

Spirogyra

sp .. Ca concentration in the water. and the concentration of Ca in

Spirogyra

sp. measured at the beginning and at the end of the study. [image:13.1496.47.1441.121.1390.2]

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Optimum light intensity (500-750 ft cd)

Maximum light in tensity (> 750 ft cd)

Minimum light intensity « 500 ft cd)

Figure 7. Various kind of light intensity treatment on Spirogyra sp. cultivation

Experiment 3. Treatment of selecte d

Spirogyra

sp. cultivation In absorbing calcium

(CaC") with different Irradiation time

Based on result of Experiment

2,

the best treatment in Ca uptake by Spirogyra sp. is 0.5 mLCaCI2/ L media in optimum light intensity (500-750 ft cd). So, in third experiment will be done by using differen t irradiation time.

The study was conducted through experiments on a laboratory scale. A total of 1.5 g of Spirogyra sp. cultured on growth media were prepared. Calcium enrichment is done by adding 0.5 mL of CaCI2 solution (36.76 g CaCi2/L distilled water) at each 1 L of growth media Spirogyra sp. (Andersen et a/. 2005).

Conta iner jar experiments put on a shelf equipped aeration. The light source of the light given by the average light intensity 705.67 ft cd . This refers to the range of optimum light for growth at 500-750 ft cd for Chlorophyta (Ryther 1965). Treatment in this study was difference while irradiation on cultured Spirogyra sp. There are two

treatments, namely irradiation time

12

hours/day and duration of exposure

24

hours/day. Each treatment performed three replications . Measurement of pH, temperature, and light intensity were done daily for seven days of culture. Weight of

Spirogyra sp. Ca concentration in water (culture medium), and the concentration of Ca in Spirogyra sp. measured at the beginning and at the end of the study. Microscopic observation of the cells Spirogyra sp. also performed at the beginning and a t the end of the study.

[image:14.1492.32.1456.79.1953.2]

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Figure 8. Different irradiation time treatment on Spirogyra sp. 24 hours/day (left) and 12 hours/day (right)

111.3. Data Analysis

Percentage change in weight and the concentration of Co is calculated by comparing the magnitude of the changes that occurred after seven days of culture with the initial value . T-test is done to see the difference between trea tments.

[image:15.1500.30.1455.89.2229.2]

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IV. RESULT AND DISCUSSION

IV.l . Result of Experiment 1 Treatment of selected filamentous microalgae

(S pirogyra

sp.) culHvaHon in various kind of calcium (CaC!., CaSO., and CaCO,)

Calcium conc entraHon in water

Calcium (Ca) concentration in water increased at the end of observation in all treatments (Figure 9). Calcium concentration was increase 49% in control. 33% in CaC03 treatment (33%) , 16% in CaC12, and 3% in CaSO. treatment. The existence of Ca in water media trial of suspected from the addition of calcium, can also be derived

from the breakdown of Ca in existing water media .

30

--

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Ol 25 E

-

ｾ＠ OJ 20 ｾ＠ 0 3 c

.-c ₁₅

0 .-- OayO ｾ＠ 0 ｾ＠ｾ＠

c ₁₀ _{- Day 7}

OJ

u

c

0

U ₅

0

U

o

Control CaCI2 CaS04 CaC03

Treatment

Figure 9. Calcium concentration in water on Spirogyra sp. cultivation with various kind of calcium

Calcium concentraHon in filamentous algae

(Spirogyra

sp.)

Ca values in Spirogyra sp. also increased at the end of the observation in all treatments (Figure 10). The percentage increase in the Ca value of the highest to the lowest in the control (40%) , CaCl2 (38%), CaC03 (25%) , and CaSO. (6%), respectively.

[image:16.1492.46.1438.100.1908.2]

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0,08

--

-"

C> 0,07

E

-

e

_0,06

>-C>

f:> 0,05

.-n.

ｾ＠ 0,04

.-c:

.Q 0,03

-

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u

6

0,01

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8

0,00 +----'

Control CaCl2 CaS04 CaC03

[image:17.1505.26.1452.79.2189.2]

Trea tment

Figure 10. Calcium concentration in Spirogyra sp. on Spirogyra sp. cultivation with various kind of calcium

Fresh weight of filamentous algae (Spirogyra sp,)

After seven days of re tention, fresh weight Spirogyra sp, increased in all treatments (Figure 11). This indicates that Spirogyra sp. has grown. The addition of

Spirogyra sp. weight from the highest to the lowest in the CaSO. treatment (110%),

CaCI2 (66%) , CaC03 (46%) , and controls (33%) , respectively.

4,0

-

3,5

C>

--

_L 3,0

C>

.-Q) _2,5

ｾ＠ L 2.0 ｾ＠ Q) ｾ＠ - DayO

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e

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>- _{- Day 7}

C>

0 _1.0

ｾ＠ .-n. Vl 0,5 0,0

Kontrol CaCl2 CaS04 CaC03

Treatment

Figure 11. Fresh weight of Spirogyra sp. on Spirogyra sp. cultivation with various kind of calcium

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The growth of

Spirogyra

sp. allegedly influence of nutrients from fertilizers Gandasil D®. Gandasil D® provides essential nutrients that algae needs. Fertilizers Gandasil D consists of N (20%),

P20S

(15%),

K20

(15%), MgSO. (1%), and equipped with micro-nutrients such as Mn, B, Cu, Co and Zn, and vitamins for plants growth such as: Aneurine, Lactoflavine, and Nicotinic acidamide.

Based on Figure II, it is known that the of CaSO. treatments cause the higest of

Spirogyra

sp. fresh weight at the end of the study. This contrasts with the results of the presence of Ca in the water or on the

Spirogyra

sp. It was alleged that Ca is an element that is not needed in large amounts by

Spirogyra

sp. in this filamentous algae.

Water Quality (temperature, light intensity, and pH)

The temperature values during the study ranged from 19.1 to 20.7 °C, and light intensity ranged from 2120-4650 lux. The average pH values during the study ranged from 7.51 to 9.13. The pH values fluctuated during the observation. Generally, the pH value in the treatment of CaSO. has a higher pH range than other treatments. Changes in pH values during the study relates to the activity of ion transport in and out of cells. Allegedly this is also affecting the uptake of Ca by

Spirogyra

sp.

IV.2. Result of Experiment 2. Treatment of selected

Spirogyra

sp. culHvaHon with calcium (CaCb) in various kind of concentraHon and different levels of light intensity

Calcium concentraHon in water

Ca values decreased only medium water at a concentration of 0.5 CaCh treatment at the optimum light intensity (500-750 ft cd). During the seven-day retention , Ca water at the treatment decreased by 3%. This indicates that the Ca in the water has been absorbed and utilized by

Spirogyra

sp. Ca value of water in the other treatments had increased by the end of the observation (Figure 12).

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40

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_-" ₃₅

'"

E

:JO

-

ｾ＠

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-

a ₂₅

ｾ＠

c

.- ₂₀

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ｾ＠

-

c

OJ ₁₀ _{. Day7}

U

c

0

U 5

a

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0

0,5 I 2 0,5 I 0,5 I

milL ml/l milL m l/l mill mllL

500 - 750 II cd > 750 II cd <500 II cd

[image:19.1492.46.1451.74.1902.2]

Treatment

Figure

12.

Calcium concentration in water on Spirogyra sp. cultivation with various kind of concentration and different levels of light intensity

Based on the concentration of Ca is added, it can be seen that the CaCI2 concentration and 1 mL/L and

2

mL/L Ca concentration increased value for all levels of

light intensity. The highest increase in Ca concentration at the end of the observations found in the treatment concentration of

2

mL/L with low light intensity <500 ft cd. It is suspected that the added Ca Ca exceeds the amount required by Spirogyra sp. so no further use. Low light intensity is also thought to reduce the ability of Spirogyra sp. in the process of photosynthesis and uptake of Ca. An increase in Ca in water treatment studies in other media besides allegedly derived from the addition of calcium, can also be derived from the breakdown of existing water Ca media research. This causes Ca are in excess amount.

Calcium concentration In filamentous algae (Spirogyra sp.)

Ca values in Spirogyra sp. at the end of the observation has decreased almost in all treatments. Increased value addition of Ca found in the treatment of 0.5 ml CaCi2/L at optimum light (500-750 ft cd) and minimum light (500 ft cd) as well as the addition of

2

ml CaCI2/L at minimum light. with the percentage increase in the value of Ca 1

%,

15%, and 19%, respectively.

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900

---' 800

(})

E ₇₀₀

-

₀

-

>- ₆₀₀

(})

0

-.-_n.

500

'"

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.-c ₄₀₀

0

""

0

300

-ｾ＠

c

11>

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c

0

u

0 100 U

a

D 0,5 1 2

a

0,5 1 2 D 0,5 1 2

ml/l mill mlIL mill ml/l mill ml/l mill mill ml/l mI/l

500 - 750 ft cd > 750 ft cd

I

<500 ft cd

Trea tme nt

I

• Day 0

. Day7

Figure 13. Calcium concentration in Spirogyra sp. on Spirogyra sp. cultivation with various kind of concentration and different levels of light intensity

Fresh weight of filamentous a lgae

(S pirogyra

sp.)

After seven days of retention , weight Spirogyra sp. increased in almost all treatments (Figure 14). This indicates that there is a growth in Spirogyra sp. were tested

in the study. The highest addition of weight Spirogyra sp. was found in the treatment of the addition of 0.5 milL at the optimum light intensity (500-750 ft cd) and followed by the maximum light intensity (> 750 ft cd) , with the addition of weights of the both treatments

46.3

and

44.3%,

respectively.

Based on Figure 14 also obtained information that the weight of Spirogyra sp. an increase in the optimum light intensity (500-750 ft cd) and maximum (750 ft cd ), whereas the minimum light intensity

«500

ft cd) tend to decrease the weight of the algae. This is allegedly due to differences in the availability of light required by Spirog yra

sp. in the process of photosynthesis, which in turn affect the growth of filamen ts in

Spirogyra sp.

[image:20.1492.26.1446.112.1810.2]

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6

-_<J) 5

-.s:

.'" 4 _Q)

ｾ＠

.s: 3

1!

ｾ＠

is; 2

ｾ＠

.-a.

on I

o

milL

0,5 I 2 0

milL mL/L milL milL

500 -750 H cd

0,5 I 2

mill ,mL/L milL

> 750 H c d

Trea tment

o

milL

0,5

ｾＯｌ＠

I 2

ml/L milL

<500 H cd

. 0ayO

. Oay7

Figure 14, Fresh weight of

Spirogyra

sp, on

Spirogyra

sp. cultivation with various kind of concentration and different levels of light intensity

Water Quality (temperature, light Intensity, and pH)

Range of pH values measured during the study are presented in Table 1. Table

1,

Range of pH values during the study

Light intensity

500 - 750 It cd

> 750 It cd

<

500

ft cd

CaC i2

Concentration

°

ml/l 0,5 mill

1 ml/l

2

mill

°

ml/l 0,5 mill

1

ml/l

2

ml/l

°

mill 0,5 mill

1 ml/l 2 ml/l

pH range 7,21-8,61 7,09-8,98 7,13-8,55 7,16-8,98 7,25-8,64 7,36-8,85 7,32-8.73 7,28-8,56 7,03-8,27 7,17-7,97 7,24-8,40 7,22-7,86

The pH values fluctuated during the observation, Results of research conducted by Sulfahri and Wulanmanuhara (2013) provide infollnation that the pH value of the culture of Spirogyra sp. with the addition of fertilizer Gandasil D® changes during 40 days of retention. Changes in pH values during the study relates to the activity of ion

[image:21.1492.36.1457.84.2075.2]

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transport in and out 01 cells. Allegedly this is also affecting the uptake 01 Ca by

Spirogyra sp. Range 01 temperature measured are presented in Table 2.

Table 2. Range 01 temperature during the study

Light intensity CaCI2 Temperature

concentration range (0C)

500 - 750 It cd

°

_mill 23,3-25,3

0,5 mill 23,2-25,5 1 ml/l 22,7-25,6 2 mill 23,4-25,0

>

750 It cd

°

_mill 23,0-25,5 0,5 mill 22,4-24,8 1 ml/l 23,0-24,9 2 mill 22,6-24,8

< 500 II cd

°

_mill 21.2-25,3

0,5 mill 22,4-25.7 1 ml/l 22,1-25,8 2 mill 22,3-26,3

The average value 01 the range 01 light intensity at the optimum light treatment

(500-750 II cd) 01616-674 It cd, maximum light treatment

(>

750 It cd) 01852-990 It cd,

and at minimum light treatment «500 It cd) at 39-73 It cd. The average value 01 the intensity 01 light is presented in Figure 15.

1200

1000

-"0

U ₈₀₀

--

-.c

.-

_ｾ＠

600 . 500 - 750 It cd

c <1>

-

_c

.>

750 It cd

.

-.r:. ₄₀₀ _<₅₀₀_It_cd

01

.-_--'

200

o

1 2 3 4 ₅ ₆ 7

Day of observation

Figure 15. Value 01 light intensity during the study

[image:22.1492.51.1449.120.2288.2]

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Spirogyra sp. cells CondlHons During the Study

Figure 16. Conditions of cells at the start of treatment (Day 0)

Conditions of cells at the end of treatment (magnification

lOx

40) a. Optimum light treatment (500-750

It

cd)

--1

[image:23.1492.48.1449.83.2201.2]

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b. Maximum light treatment (> 750 It cd)

....

-. I

o

mill CaC!' 0,5 mill CaC!'

--n

1 mill CaCI2 2 mill CaCl2

c. Minimum light treatment «500 It cd)

,.

...

...

o

mill CaCI2 0,5 mill CaC!'

...

1 mill CaCI2 2 mill CaCI2

Figure 17. Conditions

01

cells at the end

01

treatment (Day 7) [image:24.1492.51.1475.74.2089.2]

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IV.3. Result of Experiment 3. Treatment of selected

Spirogyra

sp. cultivation In absorbing

calcium (CaCh) with different Irradiation time

Calcium concentration In water

Ca concentration in water (culture medium) increased at the end of the observation, both in the period of irradiation treatment 12 hours/day and 24 hours/day

(Figure 18).

40

-

35

-

_•

ｾ＠

OJ

E 30

-

_ｾ＠

QJ

-

0 25

3 c

.-c ₂₀ 0

.-

_{- DayO}

-

0

ｾ＠

15

-

c _ Day7

QJ U

c ₁₀ 0

u

0

U ₅

O +

-12 hours/day 24 hours/day

Treatment

Figure 18. Calcium concentration in water on Spirogyra sp. cultivation in absorbing calcium (CaCb) with different irradiation time

Treatment of 24 hour exposure period increased calcium concentration by 59%, while the 12 hour exposure period increased 51%. There was no significant d ifference between treatments (P=O.4804). The presence of calcium in water media trial alleged besides comes from the addition of calcium (CaCb), can also be derived from the breakdown of water Ca contained in the culture medium.

Calcium concentration In filamentous algae

(Spirogyra

sp.)

Ca concentration in Spirogyra sp. also increased at the end of the observation in all treatments (Figure 19). Spirogyra sp. while irradiation in the treatment o f 12 hours/day is thought to have relatively better ability to absorb Ca than long irradiatio n treatment 24 hours/day.

[image:25.1492.45.1447.140.1791.2]

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450.0

--

• 400.0 --' 0) E 350.0

-

a

ｾ＠

>-0) _300.0

0 ｾ＠

.-Q. 250.0 V) c - DayO .-c 200.0 0

.--

a

- Day7

ｾ＠ 150.0

-

c OJ u 100.0 c 0 u

a

50.0

U

0.0

+----12 hours/day 24 hours/day

Treatment

Figure 19. Calcium concentration in

Spirogyra

sp. on

Spirogyra

sp. cultivation in absorbing calcium (CaCb) with different irradiation time

The concentration of Co in the treatment of irradiation time 12 hours/day increased by 23.4%. Value percentage increase is slightly higher than the old treatment of radiation 24 hours/day. with an increase of 21.8%. Based on the t test is known that

between treatments did not show significant differences (P=0.8086).

Fresh weight of filamentous algae (Spirogyra sp.)

Weights Spirogyra sp. have additional on all treatments. after culture for seven days. This indicates that the growth in

Spirogyra

sp. There are significant differences between treatments (P=0.OO21). The addition of weights Spirogyra sp. at long irradiation treatment 24 hours/day is greater than the long irradiation treatment 12 hours/day (Figure 20). The growth of

Spirogyra

sp. thought to occur due to the influence of nutrients in the growing medium that provides essential nutrients that algae needs in general.

Based on the microscopic observation of the cells Spirogyra sp .. during the seven days of culture. there are known changes in the composition of

Spirogyra

sp. Chloroplasts. Chloroplasts at the beginning of treatment (day 0) arranged in neat form spiral ribbons with clearly pirenoid composition. The composition of the chloroplast began to thin on day 4 to day 7 in the long irradiation treatment 12 hours/day. but still spiral ribbons neatly arranged (Figure 21). [image:26.1492.74.1427.113.2273.2]

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Different results found in the longer irradiation treatment 24 hours/day. The composition of the chloroplast began to thin on day 2 to day 7 and ribbons irregular spiral began on day 3 (Figure 22). It is suspected that the growth process while irradiation treatment 24 hours/day go faster due to irradiation occurs continuously. This rapid growth is also marked by the addition of weights Spirogyra sp. while irradiation in the treatment of 24 hours/day which is much larger than the treatment 12 hours/day.

6 5

-

OJ

-.r: 4 OJ

.-<1J ｾ＠ .r: 3 ｾ＠ <1J ｾ＠ - DayO

-

0 ｾ＠

>-2 - Day7

OJ 0 ｾ＠

.-n.

V) 1

0 1

-12 hours/day 24 hours/day Irradiation period

Figure 20. Fresh weight of Spirogyra sp. on Spirogyra sp. cultivation in absorbing calcium (CaCh) with different irradiation time

Water qua lity condlHons

pH values during the study ranged from 7.66 to 8.92 for radiation period 12 hours on light conditions and 7.57 to 8.74 in dark conditions, whereas the 24-hour exposure period pH values tend to be higher, with a range of values 7.72 -9.01. An increase in the pH value is presumably due to the utilization of inorganic carbon in the

fortn of C02 by Spirogyra sp. Generally, the pH had a significant increase in the algae culture.

Temperature values during the study period ranged 19,7-22.1oC 12 hours of irradiation on the light conditions and 19,3-2O,l"C in dark conditions. whereas the 24-hour exposure period temperature values tend to be higher, ranging from 2O,7-22,1°C. This is presumably related to differences in the quantity of heat from the light received

by the culture medium.

[image:27.1492.39.1446.104.1537.2]

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," ,

'P',

Treatment da

ＧＮＮｃｏｾ｟

］｟ｴＭＭＭＭ［［［［［［［［［ＺＺ］

ｔＺｲ･Ｚ｡］ｴｭＮＡＮＭＧＭ］･ＧＡｃｮＡＮＡＮｴ＠

ｾ､ｾ｡ＺＺＺｉ］］］ＺＺ［Ｍ

Treatment da 2 Treatment da '-.:3"-_ _ _

•

I . 7 I

I "1 •

Treatment da 4 Treatment da '-.:5"-_ _ _

I ' . 4

•

[image:28.1506.36.1457.33.2197.2]

Treatment da '-.:6"-_ _ _ Treatment da '-'7'--_ _ _

Figure 21. Photo cell

Spirogyra

sp. light treatment 12 hours/day

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1-' 1

•

Treatment do 0

I ' • . ,

Treatment do 2

I - . t

Treatment do 4

•

! f t . I •

Treatment do 6

Treatment do _1:... _ _ _

Treatment do 3

-=---I ' . I

Treatment do ....:5"--_ _ _

•

t Pm t

• •

[image:29.1492.34.1449.52.2088.2]

Treatment do ....,7'---_ _ _

Figure 22. Photo cell Spirogyra sp. light treatment 24 hours / day

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IV.4. Discussion

Spirogyra sp. contains a variety of minerals such as Co, Mg, Fe, Zn, and Cu. Co is the highest concentration of minerals found in Spirogyra sp., but the concentration is still much smaller than the Co concentration found in Spirulina (Rutikanga 2011). Ca enrichment cultures Spirogyra sp. expected to be an attempt to increase the concentration of Ca in the biomass Spirogyra sp.

The pH values fluctuated during the observation. Results of research conducted by Sulfahri and Wulanmanuhara (2013) provide inforlTlation that the pH value in cultures of Spirogyra sp. with the addition of fertilizer Gandasil D® changes during the 4O-day retention. Changes in pH values during the study allegedly associated with the activity of ion transport in and out of cells. It is also suspected to affect the uptake of Ca by Spirogyra sp.

Spirogyra sp. including filamentous algae are autotrophs organisms with faster growth cycle within a few days (Salim 2012). Furthermore. Salim (2012) explains that Spirogyra sp. also able to grow heterotrophic in dark conditions in enrichment cultures with cassava starch (cassava starch hydrolyzate/CSH) as the carbon source. This

process is known as mixotrophy (Eshaq et al. 2010). However. it seems in this study. the process is not dominant because mixotrophy no other than carbon. the weight of the long irradiation treatment 12 hours/day was also not increased as dramatically as in the treatment of irradiation time 24 hours/day.

Spirogyra sp. treated with irradiation time 24 hours/day had a greater weight gain when compared to longer irradiation treatment 12 hours/day. Continuous irradiation of the culture of Spirogyra sp. allegedly can stimulate the growth of Spirogyra sp. that. especially given the light is at the optimum light conditions for the growth of Chlorophyta (Rhyther 1965) .

Increased Ca concentration more on the long irradiation treatment 12 hours/day not directly proportional to the increase in weight of Spirogyra sp. during the seven days of culture. Suspected that Ca is an element that is not needed in large amounts by Spirogyra sp. Inoue et al. (2002) reported that only intracellular C02· plays a role in cell growth at the end of Spirogyra sp.

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V. CONCLUSION AND RECOMMENDATION

5.1

Conclusion

CaCI2 is the most effective type of calcium that absorbed by Spirogyra sp. The best CaCh concentration added in Spirogyra cultivation was 0.5 mL/L medium at the optimum light intensity (500-750 ft cd). Ca absorption by Spirogyra sp. more effective at irradiation time 12 hours/day. Long exposures are directly proportional to the increase in weight of Spirogyra sp. Ca is an element that is not needed in large amounts by Spirogyra sp. in their growth process.

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Acknowledgement

The author extends deep gratitude to The Osaka Gas Foundation of International Cultural Exchange (OGFICE) for financial support to this research. The acknowledgement is also offered to the Center for Environmental Research, Bogor Agricultural University for the opportunity in this research collaboration.

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