The Ability of Gracilaria Sp . to Absorb Ammonia ( NH 3N ) and its Effect on Chlorophyll Content and Growth

One of the most commonly found component of domestic waste in coastal areas is ammonia. Bioremediation techniques can be done to reduce ammonia concentration, including the use of macroalgae Gracilaria sp., as in this study. This study aimed to measure the optimum ability of Gracilaria sp. in absorbing ammonia and its effect towards the primary productivity of water exposed to ammonia. The experiment was done for 120 hours in room temperature with three replicates for each treatment. Parameter being measured was comparison of reduction of ammonia in media seawater, and the ammonia concentration in Gracilaria sp. The results of ammonia reduction by macroalgae in treatment A, B, C and D were 41.21%, 50.27%, 47.37% and 43.05% respectively. The initial concentration results of ammonia in Gracilaria sp. was 20.75 mg/kg and the final concentrations in treatment A, B, C and D were 21.08 mg/kg, 21.20 mg/kg, 22.87 mg/kg and 24.44 mg/kg respectively. The chlorophyll-a concentration in water coloumn ranged between 0.015 0.027 mg/l.


Introduction
Indonesian citizens who live in coastal areas are about 60 %, and its population continue to increase, pressuring the coastal natural resources such as beach degradation and waste disposal to the sea.Waste pollution, especially domestic waste in coastal areas is a serious problem.Domestic activities such as household activities that could be very complex have the potential to pollute coastal and marine ecosystems.Increased household activities in coastal areas cause greater volume and more types of waste produced from time to time.As a result, the burden on water bodies that have been used to dispose waste becomes too much that would eventually harm humans and the environment.
One of the most commonly found component in household waste is ammonia.The source of ammonia in water is from the breakdown of organic and inorganic nitrogen.Organic nitrogen comes from protein, urea and the decomposition of organic materials by microbes and fungi in dead plants and marine biota.Under anaerobic conditions, high concentrations of ammonia in water is toxic and it contributes to the occurence of eutrophication.Eutrophication can cause blooming of certain algae that will block sunlight from entering water and disrupt photosynthesis.This condition can cause the concentration of dissolved oxygen in the water to decrease.If dissolved oxygen levels in water bodies decreases, may not directly kill an organism, but may significantly increase its susceptibility to other environmental stresses and diseases.(Wilson 2010).
One of the methods to reduce ammonia in water is by using bioremediation techniques.Several marine organisme can be used as an agent of bioremediation in absorb hydrocarbon and organic waste from coastal area (Syakti, 2013).One of the organism that is efficient and has the potential to be used as a bioremediation agent is macroalgae.
bioremediation that uses macroalgae has the advantage of utilizing its traits, which is bioextraction and biodegradation (Black, 1995).
Bioextraction is where the thallus walls of Gracilaria sp.absorb and store nitrogen in its cells which would then be degraded with the help of photosynthesis to form energy and cells as a reflection of the macroalgae clusters's growth (Boyajian & Carriera, 1997) (Burken & Schnoor, 1997).
Examples of nitrogen absorbed by macroalgae in the form of inorganic nitrogen are nitrate and ammonia.
Gracilaria verrucosa extract is similar to immunostimulant molecules which can improve survival and growth of shrimp (Jasmanindar et al., 2018).Macroalgae is a component of autotrophs that can produce their own oxygen, where oxygen is the result of photosynthesis.The results of photosynthesis carried out by plants which have chlorophylls are called primary productivity.Photosynthesis plays a very important role in regulating the metabolism of communities living in aquatic environment.In marine ecosystems, the dominant group which photosynthesis is algae.Macroalgae is among the most productive, for example kelp forests in the Indian Ocean have a primary productivity value of 2000 g c/ m 2 / year.

Tools and materials
The materials which are used include Gracilaria sp. that is obtained from cultivation at Karangsong Beach, Indramayu; seawater as a medium for the experiment and ammonia solution as pollutant.The materials used to measure ammonia concentration are aquades, ZnSO 4 , NaOH, tartate and nessler solution.
The tools used to test the absorption capabilities of ammonia is an aquarium with a capacity of 4 liters.The tools that are used to measure water quality: thermometer, refractometer, pH meter and DO meter; to measure ammonia concentration: a UV-Vis spectrophotometer; and to measure primary productivity: Winkler bottles.

Method
The research method used was an experimental method using a Completely Randomized Design (CRD) which consists of 4 treatments with 3 repetitions, which are presented in Tabel 1.The study was conducted for 120 hours.The physical-chemical parameters observed in this study were used as supporting data to determine the absorption pattern of ammonia and to determine its effect on the growth of Gracilaria sp.Here are the physicalchemical parameters measured during the observation: temperature, salinity, DO and pH.The primary productivity was observed using oxygen method (dark bottle-bright bottle) with incubation for 6 hours.To figure out the gross primary productivity, it is calculated using the formula (Umaly and Culvin,1998) :

Physical and chemical parameters
The physical-chemical parameters observed in this research were used as supporting data to determine the absorption pattern of ammonia as well as determine its effect on the growth of Gracilaria sp.The following are physicalchemical parameters measured during the observation: temperature, salinity, DO and pH.The physical and chemical parameters measurement on the experiment's media was carried out after the addition of ammonia and observations were carried out for 120 hours.
The measurement results of the physicalchemical parameters are presented in Table 2.
The temperature experienced fluctuation during the research, but the values did not vary much among all treatments, which ranged between 23.9 -25 °C.The temperature measurement results in the study are still in a safe range for the growth of Gracilaria sp.According to Anggadiredja et al. (2006), the optimum water temperature for the growth of Gracilaria sp. is around 20 -30 ℃ .
The salinity also experienced fluctuation during the research as it is affected by the enviromental's temperature concentration (Ihsan, et al., 2015).It ranges between 25 -27 ppt and is still in a suitable range for the growth of Gracilaria sp.The acidity level in the study showed that the average values are between 7.5 to 8.2.The results obtained tend to be base.This happens due to the release of oxygen into the water from the product of photosynthesis by macroalgae which utilized CO 2 , hence reducing the availability of CO 2 (Rukmi, et al., 2012).The pH value obtained is still tolerated for the growth of Gracilaria sp.According to Aslan (1998), Gracilaria sp.grows well in a pH range of 6.0 -9.0 and has an optimum growth at pH 8.2 -8.7.
The dissolved oxygen concentration during the research ranged from 6.2 -8.2 mg/l.It is still within the safe limit for macroalgae growth.According to Aslan (1998), the optimum limit of dissolved oxygen for macroalgae growth is more than 4 mg/l.The results for DO concentrations in the 120 hours observation has a decreasing pattern.This is caused by an increase in oxygen consumption by macroalgae for respiration.Dissolved Oxygen requirements for maintenance of health and reproduction differ for different fish and invertebrate species.Also, dissolved oxygen is the important element for respiration process and decomposition by macroalgae.(Wilson, 2010).
The highest average concentration of dissolved oxygen is at T0 with a value of 8.2 mg/l.This is because the sea water that was used as the experiment's medium has been aerated using aerators for 3 days.

The effectivity of Gracilaria sp. in reducing ammonia (NH 3 -N)
The largest ammonia measurement in the experiment's media with an effectivity percentage for Gracilaria sp. is owned by treatment B of 50.27 % and the smallest is 41.21 % from treatment A. The results of the initial and final concentration of ammonia in the experiment's media are presented in Table 3 .Based on Table 3, it is known that Gracilaria sp.reduces ammonia most effectively in treatment B. It is assumed that treatment B has the right ammonia concentration for the growth of Gracilaria sp., which allowed ammonia in the experiment's media to be used optimally as nutrients that supports their growth.The right ammonia concentration can maintain the osmotic pressure inside the cell so that the exchange of water and nutrients can run smoothly.These results are still lower when compared to the researcg conducted in natural conditions.Izzati's reseach (2010) obtained results which says that the ecosystem model for shrimp windu-Gracilaria can inhibit the accumulation of ammonia in aquatic ecosystems up to 69.09 %.In his study, the ammonia concentration obtained in the experiment's medium for 4 weeks without Gracilaria was 0.022 mg/l, while the medium which used Gracilaria was 0.0068 mg/l.
Treatment A has the lowest effectivity compared to others.This happened as there was no addition of ammonia in its medium, resulting in few absorption as well.In Table 3, treatments C and D have low effectivity values.It is suspected that the concentration of ammonia contained in the experiment's media is too high.
Based on Figure 1, it is shown that at the beginning of the study, Gracilaria was able to play a good role in absorbing ammonia; this can be seen from 0-h to 48-h where the declination rate of ammonia happened quickly.This occurred due to the high dissolved oxygen content from the aeration process in the beginning of the study.Dissolved oxygen requirements for maintenance of health and reproduction differ for different fish and invertebrate species.Also, dissolved oxygen is the important element for respiration process and decomposition by macroalgae.(Wilson 2010).
Gracilaria sp. has a high absorption capacity for ammonia.A section of Gracilaria sp. which only consists of thallus, shortens the transport and regulation system of foreign substances that enter the body.Based on Figure 1, ammonia fluctuation occurred at 72-h till 120-h in the experiment's media which occurred due to the decreasing concentration of dissolved oxygen.The availability of dissolved oxygen in the media is used by macroalgae for respiration.Respiration in plants is the process of taking O 2 to break down organic compounds into CO 2 , H 2 O, and energy.

Ammonia consentration in the thallus of Gracilaria sp
Ammonia measurements in Gracilaria's sp thallus found out that the initial concentration of ammonia in the thallus amounts to 20.75 mg/kg.At the end of the study, the highest average ammonia concentration is find in treatment D at 22.44 mg/kg and the lowest was from treatment A with a value of 21.08 mg/kg.Figure 2 shows that the higher the ammonia concentration is in the media, the more the ammonia concentration found in the thallus of the macroalgae.This happens because with an increase concentration added to the media, the possibility of interaction between the dissolved ammonia with the thallus tissues becomes greater; hence, so is the absorption level.Lobban and Harrison (1997) stated that the amount of nutrients that move into the cells by osmosis depends on the nutrient concentration inside and outside the cell.The number of ammonia in the media was greater than inside Gracilaria sp.Therefore, ammonia diffused freely into the cell as needed.At the end of the research, the lowest average ammonia concentration in Gracilaria sp. was from treatment A with a value of 21.08 mg/kg.This happened as there was no addition of ammonia in the treatment, hence little absorption occurs.

Protein concentratin in Gracilaria sp.
Protein measurements were carried out at the beginning of the research before the treatments, so each treatment had the same initial protein content of 1.47 % of its gross weight.At the end of the study, the largest protein concentration value was 2.90 % in treatment D and the smallest was 2.15 % in treatment A. The effect of ammonia towards protein concentration in Gracilaria sp.i s presented in Figure 3.The ability of Gracilaria sp. to absorb ammonia in water is related to the protein concentration in its thallus.Based on Figure 3, the more ammonia is given, the higher the protein value would be.This shows that there was an occurrence of ammonia absorption in the water media by Gracilaria sp. which was then converted to protein.The protein value obtained is relatively small compared to Handayani's et al. (2014) research.In his study, the protein values in red macroalgae ranged from 6 -20 % of its gross weight.

Chlorophyll-a concentration in the experiment's media
Chlorophyll-a measurements were carried out at the beginning of the research before the treatments, so each treatment had the same initial concentration of chlorophyll-a which is 0.015 mg/l.At the end of the study, the highest average chlorophyll-a was 0.027 mg/l in treatment B and the lowest was 0.018 mg/l in treatment A. The results are shown in Figure 4. , the lowest chlorophyll-a value is 0.018 mg/l in treatment A, where no ammonia was added so as to deprive nutrients from the macroalgae, resulting in inhibition of the metabolic process.High and low chlorophyll concentrations are affected by several physical-chemical parameters of water such as temperature, light, intensity and nutrients.
The measurement results of chlorophyll-a concentration ranged between 0.015 -0.027 mg/l.The range of chlorophyll-a concentrations fit into the category of moderate condition.This is based on the quality standards of the Decree of the State Minister of Environment ( 2004); Chlorophyll-a category: less than 0.015 mg/l is categorized as in good condition, 0.015 -0.03 mg/l as moderate and more than 0.03 mg/l as bad condition.The result is thought to occur due to ammonia concentrations that was too high in the media, causing the chlorophyll synthesis and photosynthetic process to be inhibited.Medium with high nutrient content can disrupt photosynthesis (Lobban & Harrison, 1997).Figure 4 shows that ammonia treatments with more than 0.5 mg/l leads to a lower chlorophyll-a concentration.It can be assumed that high ammonia concentration can disrupt the photosynthesis process which makes the chlorophyll-a concentration in the water becomes low.

Total chlorophyll in the thallus of Gracilaria sp.
The initial concentration of chlorophyll measurement was carried out at the beginning of the research before the treatments, so each treatment had the same initial chlorophyll concentration which is 0.7 mg/100g.At the end of the study, the highest average value of total chlorophyll was 1.6 mg/100g from treatment B and the lowest was 0.8 mg/100 g from treatment A. The average value of total chlorophyll in Gracilaria sp. is presented in Figure 5 .

Based on
Figure 5, the lowest chlorophylls average was 0.8 mg/100 g in treatment A, where no ammonia added to the medium, resulting a nutrient deficiency for the macro algae and disrupt its metabolic process.Ammonia is nitrogen source for the growth of macro algae as nitrogen is important for the synthesis of chlorophyll, proteins and enzymes.Whereas in treatment D, the amount of ammonia given was too high resulting a distruption in the synthesis of chlorophyll and photosynthesis process.

The weight growth of Gracilaria sp.
Weight measurement was carried out before the treatment began, so each treatment has the same initial weight of 14 grams.At the end of the research, the largest average weight was from treatment B with a value of 14.15 grams and the lowest was 14.06 grams from treatment D. The weight growth results of Gracilaria sp. are presented in Figure 6.Based on Figure 6, it is known that treatment B has the highest average growth weight, where the ammonia concentration is the right concentration for the growth of Gracilaria sp.In treatment B, Gracilaria sp. was able to grow and develop well as well as able to utilize ammonic in the medium as a nutrient source, i.e. as a source of nitrogen.Lobban & Harrison (1997)  Based on Figure 6, treatment C and D produced a generally low growth of biomass for the macroalgae.This occurred because the concentration of ammonia in the media was not appropriate for the growth of Gracilaria sp.According to the Decree of the Minister of Environment ( 2004), the standard quality of ammonia (NH 3 -N) for marine biota is 0.3 mg/l.Besides nutrients, macro algae growth is affected by other several factors, such as light (Hasan, Rejeki, & Wisnu, 2015).At the time of the research, the lighting in the room was not evenly distributed which caused a reduction in the amount of light received and caused a disrupt in photosynthesis.If photosynthesis is disrupted, macroalgae cannot grow optimally.Other than the lack of light, it may also happen due to poor water movement in the media.The water parameters that support the growth of Graciliaria sp.seaweed include adequate nutrients and relatively stable water movement where mixing process occurs, thus helping macroalgae to absorb nutrients well and increase its growth (Putra, Aryawati, & Isnaini, 2013).

Ammonia bio-concentration factor in Gracilaria sp.
The bio concentration factor of ammonia is a useful parameter to determine the ability level of organisms or living creatures to absorb ammonia.The results of the ammonia bio-concentration factor are presented in Figure 7. Based on Figure 7, the highest BCF value is 23.5 from treatment B and the lowest is 20.4 from treatment A. The values show that Gracilaria sp. has a high accumulation level for ammonia.This is based on 3 BCF categories of plants which are divided into accumulators, excluders and indicators.The accumulator has a BCF value of > 1, excluder has a BCF value of < 1 and an indicator plant has a BCF value close to 1 (Susana & Suswanti, 2013).Boyajian & Carriera (1997) stated that Gracilaria sp., with its phytoextraction properties, can accumulate and store organic matter such as nitrogen in the form of ammonia in thallus cells .

Conclusion
Based on the research results and discussion, it can be concluded that: 1. Gracilaria sp has the ability as a bioemediation agent to reduce ammonia concentration in water with a consequence of interference towards the growth of Gracilaria sp.The higher the concentration of ammonia, the lower the growth of Gracilaria sp. 2. The chlorophyll concentration decreases with increasing ammonia concentration

Figure 1 .
Figure 1.Ammonia concentration graph of the experiment's media

Figure 2 .
Figure 2. Average value graph of ammonia concentration in Gracilaria sp.

Figure 4 .
Figure 4. Average Concentration Graph of Chlorophyll-a in Media

Figure 5 .
Figure 5 .The average value graph of total chlorophyll in Gracilaria sp.

Figure 6 .
Figure 6.The average weight value graph of Gracilaria sp.
stated that at the right nutrient concentration, the osmotic pressure in the cell does not change rapidly which allows the exchange of water and 0smoothly, and a good metabolic process leads to optimum growth.These nutrients will increase the activity of cell metabolisms by entering cells little by little and then developing vacuoles that are inside the cell.Vacuoles play a very important role in life because the plant's survival mechanism depends on the ability of vacuoles to maintain the concentration of dissolved substances in them.Plants grown in nutrient rich conditions will deposit large quantities of such nutrients in vacuoles of vegetative tissues, this allows the plant to survive subsequent periods of nutrient deficiency by mobilizing the vacuolar store.Thus, the solute composition of vacuoles is highly dynamic and reflects changes in the environment and developmental stage(Isayenkov et al., 2010).

Figure 7 .
Figure 7.The value of ammonia bio-concentration factor in Gracilaria sp.
Ability of Gracilaria Sp. to Absorb Ammonia

Table 1 .
Treatment Concentration of Ammonia and Gracilaria sp.

Table 2 .
The Measurement Results of Physical and Chemical Parameters Parameter Range of the research results Optimum range for the growth of Gracilaria sp.

Table 3 .
The effectivity of ammonia decrease by Gracilaria sp.