The Effect of Salinity on the Biomass of AVICENNIA MARINA and

RHIZOPHORA MUCRONATE Grown at Reed Bed System Reactor

with Continuous Flow

Tri Cahyo Puspaningrum

1,a

and Harmin Sulistiyaning Titah

Department of Environmental Engineering, Faculty of Civil, Planning and Geo Engineering

Institut Teknologi Sepuluh Nopember, Indonesia

Keywords: Growth, Mangrove, Reed Bed System.

Abstract: Salinity is one of the environmental factors having an important role in controlling mangrove growth. Each

type of mangrove has different adaptability. This condition causes the differences of structure and

composition of mangrove with distinctive boundaries, ranging from zones close to land to zones close to the

ocean. This research was aimed to examine the ability to grow mangrove (Avicennia marina and Rhizophora

mucronata) at various levels of salinity, using reed bed system reactor with continuous flow and the addition

of the bacteria Vibrio alginolyticus. The reactors were arranged in series system, namely reactor with

Avicennia marina (AM), reactor with Rhizophora mucronata (RM), reactor with Avicennia marina and

bacteria of Vibrio alginolyticus (AMVA), and reactor with Rhizophora mucronataand Vibrio

alginolyticus(RMVA). The artificial salinity that was used i.e 20 ‰ and 25 ‰. Physical observation of the

mangrove growth indicators was conducted during the exposure time. The fresh weight (FW) and dry weight

(DW) were measured at day 0 and the last day of experiment. The monitoring parameters such as pH and

temperature were also measured. The results showed the FW and DW increased in all reactors. Avicennia

marina with added bacteria had the greatest growth at the salinity concentration of 25‰ with 69,27 g of DW.

Salinity of 25‰ showed a greater growth result than salinity with a concentration of 20‰.

1 INTRODUCTION

Salinity is the level of salinity or dissolved salt

contained in water in grams per litre of seawater

(Chimayati and Titah, 2019). Salinity is one of the

defining environmental features of mangrove habitats

and ranges from seasonally freshwater to hypersaline

conditions (Flowers and Colmer, 2008). Salinity can

be interpreted as a condition where salt dissolves

excessively and causes bad conditions for plant

growth (Syakir et al., 2008). According to Bengen

(2003) salinity greatly determines the development of

mangrove area, this can occur because of the

influence of salinity which can divide mangrove

growth areas into several zones, from the nearest

zonation or bordering the sea (proximal zone) to the

farthest zonation from the sea (distal zone).

According to Purwanti et al. (2006), classification of

the sample water for salinity parameters is divided

into freshwater with a value of <0.5‰, brine water

with the salinity ranging from 0,5–30‰, salty water

30–50‰ and very salty water or sea water, which has

a salinity of more than 50‰. Mangrove has the ability

to tolerate the sea salinity and grow at above average

levels (Ananthakrishnan, 1982; Flowers et al.,1977).

Mangrove forest ecosystems are often called brackish

forests because they are located in brackish areas,

which are areas with salinity or salinity between

0,5‰ and 30‰. Another name is the tidal ecosystem

because it is located in areas affected by tides

(Indriyanto, 2006). They adapt themselves to

fluctuating environment in several ways such as salt

exclusion from roots (Hegemayer, 1997), salt

secretion (Fitzgerald et al., 1992) and accumulating

organic acids as osmotica to counter toxic effects of

salinity (Popp, 1984). Mangrove plants comprise a

heterogeneous group of independently derived

lineages that are defined ecologically by their location

in upper intertidal zones of tropical and sub-tropical

climates and physiologically by their ability to

withstand high concentrations of salt or low levels of

soil aeration (Basyuni et al., 2007).

Puspaningrum, T. and Titah, H.

The Effect of Salinity on the Biomass of AVICENNIA MARINA and RHIZOPHORA MUCRONATE Grown at Reed Bed System Reactor with Continuous Flow.

DOI: 10.5220/0010864200003261

In Proceedings of the 4th International Conference on Marine Technology (senta 2019) - Transforming Maritime Technology for Fair and Sustainable Development in the Era of Industrial

Revolution 4.0, pages 30-37

ISBN: 978-989-758-557-9; ISSN: 2795-4579

The flora community found in mangrove forests

has undergone adaptation and specialization as a

mechanism for living in an environment with high

levels of salt (Kustanti, 2011). Mangrove can adapt to

low oxygen levels, tolerate high salt levels and can

adapt to unstable soils and the influence of tides

(Bengen, 2003). According to Saparinto (2007),

mangroves depend on seawater (tides), freshwater,

and sediment as a source of nutrients. In high salt

conditions, plants will face two problems: obtaining

water from negative potential groundwater and

overcoming the high ion concentration of sodium,

carbonate, and chloride which may be toxic

(Salisbury and Ross, 1995). One indicator of

mangrove growth is it is physically influenced by the

sediment where it lives, which contains macro and

micronutrients, oxygen, and fresh water to maintain a

balanced salt content (Chrisyariati et al., 2014).

Limiting factors for mangrove production and growth

include temperature and sunlight, salinity, anoxia and

tides, bioturbation, and nutrient availability (Alongi,

1998). The growth and physiological mechanisms of

mangroves differ in nature due to their complexity of

structure and differences flooding regime, tidal

inundation, a rapid influx of extra nutrients as well as

the type of soil (Naidoo, 1987).

Avicennia has the ability to tolerate a wide range

of salinity. The species is able to grow well in salinity

up to 90 ‰ but at extreme salinity, the tree grows

stunted and the ability to produce fruit is lost (Noor et

al., 2006). Avicennia marina collects the highest ion

concentration from Rhizophora mucronata

(Scholander et al., 1962), which means that the ability

of Rhizophora mucronata to accumulate inorganic

ions is lower than that of Avicennia marina (Titah et

al., 2019).

The influence of salinity on mangrove growth was

reported by Clough (1984) who stated that the highest

number of Avicennia marina and Rhizophora stylosa

dry weight was obtained when grown at 25‰

seawater content. He also reported that CI- and Na+

ion levels were greater than K+, Ca2+, and Mg2+

ions in mangrove plant roots, stems and leaves which

grown in five different concentrations of seawater

that he tried. Stem and Voigt (1959) in Tomlinson

(1986) argue that it was better to use low level of

seawater for breeding Rhizophora. Connor (1969) in

Tomlinson (1986) found the optimum conditions for

Avicennia marina growth was in a solution containing

50‰ Na+ ions and Na from seawater.

Bacteria can increase plant tolerance to

environmental conditions that might reduce plant

growth or development (Sulastri, 2018). Vibrio

alginolyticus, a helobacterium bacteria, can live in

areas with high salt levels and are resistant to

radiation and live in salt crystals. It functions in the

process of the nutrition cycle and supports the life

buffer of the ecosystem environment (Thompson et

al., 2004). Vibrio alginolyticus bacteria is indeed

found in saline water. These bacteria can grow and

live in the area of plant roots which were in water that

has a high level of salinity. Vibrio bacteria grows at

pH 4–9 and optimally at pH 6,5–8,5 or under alkaline

conditions with pH 9,0 (Chimayati and Titah, 2019).

Vibrio bacteria could die under the acidic conditions.

Kurniawan et al., (2018) reported that Vibrio

alginolyticus needed 2 h at pH 8 to grow, meanwhile

it needed 48 h at pH 5. He indicated that the bacteria

did not develop at pH below 5, shown by an Optical

Density (OD) value of 0.

Plant growth can be defined as the enhancement

size process and number of plant cells followed by the

growth of plant dry weight, while the development of

plants can be interpreted as a process towards

achieving maturity (Kolinug et al., 2014). Plant

growth and development is divided into two phases:

vegetative growth phase and the generative growth

phase (Prayunita, 2012). According to Popp (1994),

mangroves collect high concentrations of inorganic

ions like most other salt tolerant plants that function

in leaf and other tissue osmoregulation. This form of

osmoregulation involves the synthesis and

accumulation of organic compounds sufficient to

decrease the osmotic potential of cells and increase

turgor pressure (Kusumiyati et al., 2017). Flowers et

al. (1977) argue that in the early stages of adaptation

to high salinity or the increase of salinity when the

salt concentration in the liquid was increasing, the

rate of ionic absorption was related to the growth rate

of the plant. Mangrove plants take salt as nutrients for

their growth needs.

The aim of this study was to determine the effect

of salinity on the growth of mangrove Avicennia

marina and

Rhizophora mucronata with artificial

salinity variation of 20‰ and 25‰ in a reed bed

system reactor combined with Vibrio alginolyticus

bacteria.

2 MATERIALSAND METHODS

2.1 Location of Research

This research was conducted at the greenhouse of the

Department of Environmental Engineering, Institut

Teknologi Sepuluh Nopember, ITS, for the

implementation of the reed bed system reactor and at

the Environmental Remediation Laboratory in same

The Effect of Salinity on the Biomass of AVICENNIA MARINA and RHIZOPHORA MUCRONATE Grown at Reed Bed System Reactor

with Continuous Flow

department for bacterial propagation and analysis of

the growth parameters.

2.2 Material and Method

2.2.1 Bacterial Preparation

The inoculation stage used NA (Nutrient Agar)

(Merck, USA) and TCBS (Thiosulfate Citrate Bile

Salt Sucrose) (Merck, USA) for selective media. NA

media was used as the initial inoculation media.

TCBS media is a special selective media for Vibrio

alginolyticus bacteria. The selective media was used

for the breeding of bacteria. This stage was conducted

to confirm that the growing bacteria on the media was

Vibrio alginolyticus. In this study, the addition of the

inoculum Vibrio alginolyticus was 5% (v/v) in each

reactors or it reached 300 mL/reactor. The

preparation of TCBS media was conducted by

dissolving of 22,.25 g of TCBS media in 250 mL of

sterile aquadest and then it was put in a 250 mL of

erlenmeyer. Around 8 g of NB was used for

preparation media. Before the media was dissolved

with the aquadest, the aquadest must be sterilized

using a autoclave (Hirayama, Japan). The media

dissolving was conducted using a stirring rod on a

heating stove until the media boiled. After that, the

media was poured into an aseptic sterile petri dish.

After the media thickened, the petri dish was turned,

then the media was stored in the refrigerator. The

regrowth of Vibrio alginolyticus was conducted by

inoculating those bacteria on a new TCBS media

using ose. All inoculation activities must be sterile by

working near the Bunsen and ose needles must also

always be sterile. After that, the inoculating media

was put in an incubator for 24 h at 37oC. After the

growing process, the bacteria was transferred into a

sterilized NB (Nutrient Broth) media (Merck, USA)

and put in the orbital shaker KIA Japan for 2 days to

get the OD value of 1. OD measurements were carried

out using a spectrophotometer GENESYSTM 30

Visible Thermo Scientific USA. Bacteria with an OD

value of 1 meant that it was ready to be poured into

the reactor.

2.2.2 Plant Preparation

This research used two species of mangrove:

Avicennia marina and Rhizophora mucronata. All

plants were collected from the mangrove nursery in

Wonorejo, Surabaya. The age of the plants was about

3 months old. The second stage was to prepare

mangrove plants by separating each type of mangrove

and then cleaning it by washing the sludge on the

roots. Before all plants were used for research, the

plants were acclimatized for 2 weeks to determine the

ability of plants to grow on the concentrate of saline

water to be used.

2.2.3 The Artificial Saline Preparation

This research was carried out by an experimental

method and by the observation of the mangrove

condition during the operation of reactor. The saline

water used in this study had an artificial salinity. The

artificial salinity was made using distillation water

and pro-analysis NaCl powder (Merck, USA). The

pro-analysis NaCl was dissolved in distilled water.

Around 5,370 g of pro-analysis NaCl was needed to

make a salinity of 20‰, and it needed 6,712.5 g to

make a salinity of 25 ‰.

2.2.4 Reactor Preparation

The reed bed reactors in this study was constructed

from fiberglass, measuring 70 x 50 x 40 cm.

Fiberglass is a strong and anti-rust material (Sunyoto

et al., 2016). There were 12 reactors: 4 reactors with

the addition of bacteria, 4 reactors without bacteria

and 4 reactors without plants as the control. The reed

bed system reactors were then arranged in a series

arrangement with a continuous water debit of 18

mL/minute. Preparation of reed bed system reactors

in series was carried out based on the zoning of

mangrove species growth ecosystems in nature.

Figure 1 and 2 describe the reed bed system reactor.

The code of each reactor:

- AMVA 25-RMVA 25:

Avicennia marina + Vibrio alginolyticus 25‰-

Rhizophora mucronata + Vibrio alginolyticus

25‰

- AM 25-RM 25:

Avicennia marina 25‰ - Rhizophora mucronata

25‰

- BK1 25 - BK2 25:

Control Reactor 1 25‰ - Control Reactor 2 25‰

- AMVA 20 - RMVA 20:

Avicennia marina + Vibrio alginolyticus20‰ -

Rhizophora mucronata + Vibrio alginolyticus

20‰

- AM 20 - RM 20:

Avicennia marina 20‰ - Rhizophora mucronata

20‰

- BK1 20 - BK2 20:

Control Reactor 120‰ - Control Reactor 2 20‰

senta 2019 - The International Conference on Marine Technology (SENTA)

Figure 1: Reed bed system reactor with bacterial addition.

Figure 2: Reed bed system reactor without bacterial

addition.

The media composition of each reactor was as

follows: layer of gravel with a diameter of 2 cm and

a height of 5 cm. The second layer of gravel with a

diameter of 1 cm and a height 5 cm. The third layer

was fine sand with a height of 10 cm and artificial

saline water was put on the top of the filter media. The

height of the artificial saline water was 10 cm and

each reactor needed 33,5 L of the water.

Monitoring parameters also were measured.

Those parameters were pH and temperature. The pH

measurement was carried out using a portable pH

meter digital Senz pH Singapore. Temperature

measurement was conducted using OHAUS Starter

3100C Conductivity Bench USA.

The fresh weights (FW) and dry weights (DW)

were measured for each part of the sampled plants

(roots, stems, and leaves). The FW was conducted as

soon as possible after plants were cleaned using

tissue. All plant parts were put in an oven at 105°C

for 24 hours for the dry weight measurement. After

that, the total DW of all plants could be calculated.

The calculation of Plant Water Concentration

(PWC) was conducted by formula based on Penuelas

et al. (1997).

= ((

W – DW) / DW) 100 (1)

Preliminary research conducted by acclimatizing

the test biota used in this study revealed that

mangroves were able to adapt to the conditions or

environmental media of the actual experiment and

that the plants were able to adjust to the conditions of

the media used in the study.

Physical observations of mangrove plants were

carried out during the acclimatization of mangrove

plants at salinity concentrations of 20‰ and 25‰.

Acclimatization was also aimed at making plants able

to adjust to the growing environment in the treatment

(Cahyani et al., 2016). Acclimatization results

obtained showed that Avicennia marina and

Rhizophora mucronata were able to grow well at

salinity concentrations of 20‰ and 25‰.

Based on observations made for 2 weeks, there

were no significant changes on Avicennia marina

plants. The leaves and the stems of plants showed

good conditions. This indicated that Avicennia marina

plants can survive at salinity concentrations of 20‰

and 25‰. The plant did not wilt during

acclimatization, however some leaves of Avicennia

marina showed discoloration at a concentration of

25‰ without bacteria addition.

Rhizophora mucronata plants were able to

survive in concentrations of 20‰ and 25‰, although

some withering leaves occurred. Accordint to Titah et

al (2018), the salinity concentration of 30‰ can be

toxic to Rhizophora mucronata.

The range of saline temperature was 29

C - 32

(Figure 3). Mangrove and bacteria can live in this

range of temperature. Bacteria can survive, grow and

develop at certain temperature limits. Vibrio

alginolyticus can survive at optimum temperatures

between 30-35

C, while the bacteria cannot grow

below 4

C and above 45

C: Vibrio alginolyticus will

die at 55

C (Prajitno, 2005). During the experiment,

the temperatures of several reactors were similar

because the reactors were placed in same area and

sunlight reached all reactors. Measurement showed

that the water temperature was suitable for the growth

of the planted mangroves, especially for Rhizophora

sp. According to Saparinto (2007), mangrove species

Avicennia sp. grows well at temperatures between 18-

20ºC, Rhizophora sp., Ceriops sp., Excoecaria sp.,

Lumnitzera sp. at 26- 28ºC, and Bruguiera sp. at a

temperature 27ºC.

The Effect of Salinity on the Biomass of AVICENNIA MARINA and RHIZOPHORA MUCRONATE Grown at Reed Bed System Reactor

with Continuous Flow

3 RESULTS AND DISCUSSION

The parameter of pH is a measure of acidity or

basicity of a liquid. A normal pH is represented by the

value range of 6-8. The pH of water depends on the

type of the discharge of water (Fardiaz, 1993). The

value of pH at all reactors showed a neutral pH when

the reed bed reactor was run. Based on the data, the

average pH range was 5.8-8.1 (Figure 4). The pH

affected the growth rate of the bacteria. Each

organism has a different optimum pH ranges:

mangroves can survive at pH levels of 6-8.

Mangroves aged 36 months are more resistive to large

water pH range: older mangroves are known to have

greater tolerance for pH and salinity ranges because

they have a stronger root

system compared to younger

mangroves (Chrisyariati et al., 2014). The average pH

value range of this experiment was 6 to 8. This showed

that the pH level of the water was still in an acceptable

range for both the mangrove and the aquatic biota

growth. According to Koch (2001), pH level is closely

related to decomposer activity: the more acidic the

environment is for the decomposer, the slower the

decomposing process of inorganics. The slow process of

decomposition could greatly inhibit vegetation growth

Figure 3: Temperature measurement.

ure 4:

H measurement.

senta 2019 - The International Conference on Marine Technology (SENTA)

due to a lack of nutrient and mineral supply. In addition,

a pH value range of 6.0 to 6.5 can reduce the diversity

of plankton and benthic species (Effendi 2003).

The results of the growth and development

process can be observed from the fresh weight and

dry weight. Plant fresh weight is the result of the

measurement of the fresh weight of plant biomass and

the total accumulation of material produced during

the growth. Therefore, the observation of fresh plant

weight and fresh weight is needed to determine the

plant biomass (Buntoro et al., 2014). Whereas dry

weight, according to Gardner et al (1991), is the result

of the net hoarding of CO

assimilation throughout the

growing season which reflects the accumulation of

organic compounds plants have successfully

synthesized from inorganic compounds, especially

water and CO

Figure 5 shows the FW and DW of Avicennia

marina and Rhizophora mucronata during the

operation of the reed bed reactor for 18 days in

salinity of 20‰ and 25‰. Based on the figure, the

FW and DW of Avicennia marina and Rhizophora

mucronata increased. It indicated that Avicennia

marina and Rhizophora mucronata can grow

normally during the operation of a reed bed reactor.

Avicennia marina plants with the addition of

Vibrio alginolyticus at a salinity concentration of

25‰ showed the highest of FW compared to other

plants on the last day of the experiment. The FW of

Avicennia marina was 69.26 g, and the DW was

24.03 g. This DW value of Avicennia marina is the

lowest of all plants used in this experiment:

Rhizophora mucronata plants, in the same conditions,

had the highest DW value of all plants with 34.16 g

of DW and an FW value of 53.8 g. The addition of

Vibrio alginolyticus is suspected to play a role in the

uptake of nutrients such as Na dan Cl ions. According

to Westrich et al. (2016), Vibrio bacteria does play a

key role in the cycling of the essential micronutrient

Fe.

This indicates that the absorption of salinity by

Rhizophora mucronata with the addition of Vibrio

alginolyticus bacteria is very good

Based on the

results of the FW and DW calculations, the rather

stable value of FW and DW would produce a stable

water content. Figure 6 shows that the FW and DW

Figure 5: FW and DW measurement.

ure 6: Plant wate

concentration.

80



70



60



Day‐ 0

0

AMVA25‐AM25‐AMVA20‐ AM20‐ AMVA25‐ AM25‐ AMVA20‐ AM20‐ RMVA25

RM25RMVA20 RM20RMVA25 RM25RMVA20 RM20

RM20

5

AM25

RM25

0



AM20

RM20

5

RM25

0

AM20

Weight(gram)

The Effect of Salinity on the Biomass of AVICENNIA MARINA and RHIZOPHORA MUCRONATE Grown at Reed Bed System Reactor

with Continuous Flow

values of Rhizophora mucronata plant with the

addition of Vibrio algionolyticus bacteria were not

different. It indicates that the water content in the

Rhizophora mucronata plant with the addition of

Vibrio algionolyticus bacteria were stable.

Based on the calculation of water plant

concentration (WTP), on the Figure 6 and Table 1, the

value of WTP in some reactors were in that range.

However, in the reactor with Rhizophora mucronata

and Vibrio alginolyticus at a salinity of 25‰, the

WTP value was 36%.

These results are in accordance with the best WTP

value for plants (50-70%). Based on the prior

definitions of plant growth, it can be considered that

there was growth as there was an increase in FW and

accumulation of DW. A good growth of mangrove

plants is shown by the increase in DW values

(Nurdin, 2008).

The difference in DW can be caused by the

number of leaves. The leaves are a place for the

accumulation of plant photosynthesis. An increase in

the process of photosynthesis can also increase the

result of photosynthesis. The increase of

photosynthesis activity can increase the amount of

organic compounds in plant. The organic compound

could then be transported to all plant organs and affect

the dry weight of plants.

4 CONCLUSIONS

Based on the results, the level of salinity affects the

growth rate of mangrove plants. Based on the

calculation of FW and DW values, the FW of

Avicennia marina with the addition of Vibrio

alginolyticus bacteria in a salinity concentration level

of 25‰ was 69.27 g, which is highest FW value of all

other plants. The highest value of DW was obtained

in the Rhizophora mucronata plant with the addition

of Vibrio alginolyticus in a salinity concentration of

25‰, reaching 34.16 g. The Rhizophora mucronata

plant with the addition of Vibrio alginolyticus bacteria

in a salinity concentration of 25‰ had the most stable

water content value with an FW value of 53.8 g and

DW of 34.16 g, resulting in a water content value of

57.46%. In conclusion, concentration of salinity and

the addition of Vibrio alginolyticus can affect the

FW and DW of mangrove plants in a reed bed system

reactor with continous flow.

The results showed that all two mangrove

species are highly salt tolerant and can survive in

brackish water and perhaps even higher salinity

although all the studied species were under

rehabilitation condition in a mangrove conservation

center. Avicennia marina had the best tolerance to

highly saline conditions since this species maintains

very negative water potential under saline conditions.

We hope that our research will provide the

necessary groundwork for further researches, for

example in the are of bio desalination with mangrove

in reed bed system reactor with or without bacterial

addition with continuous flow.

ACKNOWLEDGEMENTS

The author would like to thank Kemenristek DIKTI

(Ministry of Research, Technology and Higher

Education, Republic of Indonesia) for the funding of

this research through the Penelitian Dasar Unggulan

Perguruan Tinggi – second year research, PDUPT

2019 scheme (Contract 5/ E1/KP.PTNBH/2019 and

No. 920/PKS/ITS/2019).

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