The Utilization of Gracilaria verrucosa as shrimp ponds wastewater biofilter

Gracilaria verrucosa as biofilter can absorb and utilize inorganic nitrogen and phosphorus contained in pollutants for its growth. This research aims to know the ability of G. verrucosa as shrimp pond waste biofilter, to know the difference in the quality of shrimp pond wastewater before and after treatment, and to learn more about the absorption capacity of G. verrucosa capability against shrimp pond wastewater. The experiment was conducted on a laboratory scale using a Completely Randomized Design (CRD) consisting of four treatments with three replications using G. verrucosa of different weights which were 100 g, 150 g, 200 g, and controls. The parameters observed in the study were water quality parameters, seaweed biomass, and seaweed absorption. The results obtained were temperature 27.2–30.1°C, TSS 7–76 mg.L, pH 7.42–8.83, salinity 16–18 ppt, DO 1.7–5.3 mg.L, biomass 74–210.7 g, ammonia effectively decreased on 10 day by 90%, nitrate on 20 day was 22.2% and phosphate value on 30 day was 20.1%. G. verrucosa absorbed nitrogen (N) 0.08% and phosphorus (P) 0.35%. G. verrucosa is potential as a biofilter and can be used as a species candidate for IMTA system.


Introduction
Shrimp farming activity in the fishpond will produce a significant amount of organic and inorganic (dissolved nitrogen and phosphorus) materials [18] which produced from residual feed waste, feces, and residual metabolism activity. The waste can change sediment composition which triggers eutrophication, and also causes water hypoxic or anoxic condition [8,16]. Aquaculture wastewater which has rich in nutrients is reported to cause blooming phytoplankton [12,17]. If blooming occurs continuously, it will threat the biodiversity and disturb the ecosystem balance.
Gracilaria is a genus of red seaweed which has been utilized in the Integrated Multitrophic Aquaculture system (IMTA system) [1,7,17]. Furthermore, Gracilaria has high economic value as raw material for the nutraceutical and pharmaceutical industry. Gracilaria verrucosa is mostly cultivated by farmers in Indonesia, especially on the northern coast of Java Island [5].
Gracilaria can absorb and extract inorganic material such as dissolves nitrogen and phosphorus [14,18] within its thallus cells for its growth. Therefore, Gracilaria is considered to increase the water quality of shrimp pond wastewater. This research is aimed to find out the ability of G. verrucosa as biofilter for shrimp pond wastewater to improve its water quality.

Location and Date of Research
This research was performed in January -April 2019 at the Center for Brackish Water Aquaculture (Balai Besar Perikanan Budidaya Air Payau) Jepara, Indonesia.

Water Quality Measurement
The water quality measurement included physical and chemical parameters. The physical parameter measured including temperature, Total Suspended Solid (TSS), and salinity. The water temperature was measured using a thermometer. Salinity was measured using a refractometer. The Total Suspended Solid (TSS) was measured using the photometric method.
The chemical parameter included dissolved Oxygen (DO), pH, nitrate, phosphate, and ammonia. pH was measured using pH meter and dissolved oxygen using the DO meter. The nitrate parameter measurement was done using method 2.6-Dimethyphenol, phosphate using Phospormolybdenum Blue method, and ammonia using salicylate method. The measured water samples were collected from all research containers. The water quality observation using physical parameters was conducted every five days, and the observation for chemical parameters was conducted every ten days.

Absorption Measurement
The absorption measurement of Nitrogen (N) and phosphorus (P) content in G. verrucosa seaweed was conducted on the 0 th and 30 th day. The measurement of N and P content in seaweed tissue was performed using AOAC 976.05.20 th Ed. 2016 SNI 2803-2012 method.

Statistical Analysis
The research was performed using the experimental method by Complete Random Design. The data result of water quality parameter measurement (nitrate, phosphate, ammonia, biomass, nitrogen, and phosphor) contents were analyzed using one-way ANOVA then followed by Tukey test analysis with the confidence level of 95%. The statistical analysis was conducted using SPSS program version 20.0. The ammonia concentration on the 0 th day was 5 mg.L -1 , and the concentration on the 10 th day was 0-1.76 mg.L -1 . The control without G. verrucosa showed increasing of ammonia concentration after the 20 th day. The ammonia concentration on the 20 th day was 0.1-0.25 mg.L -1 , and the 30 th day was 0.14-1.12 mg.L -1 . Meanwhile, the ammonia content on the 100 grams treatment on the 20 th day and 200 grams on the 30 th day were decreased because it absorbed the existing waste content. Nitrate concentration on the 0 th day was 2.9 mg.L -1 , while on the 10 th was 3.7-9.3 mg.L -1 . Nitrate concentration on the day-20 was 3.5-6 mg.L -1 , while on the 30 th day was 2.8-7.6 mg.L -1 . The nitrate absorbed by G. verrucosa occurred on the 20 th day, therefore the nitrate concentration in fishpond wastewater also decreasing. Phosphate concentration on day 0 was 0.8 mg.L -1 , 10 th day was 3-7 mg.L -1 and the 20 th day was 5-9.7 mg.L -1 . The increase of phosphate on the 10 th and 20 th day occurred on all treatments. The phosphate concentration on the 20 th was 5-9.7 mg.L -1 , while the 30 th day was 3-8 mg.L -1 . Phosphate concentration on the 10 th to 20 th day increased due to the waste decomposition process. The biomass of G. verrucosa experienced increasing on the 10 th day. After that, the biomass tended to reduce on the 20 th and 30 th day since waste nutrient content decreased.   The phosphorus content (P) in G. verrucosa tissue increased at the end of the research, from 0.72% to 0.95-1.07%. The statistical test in Table 2 does not indicate a significant difference in phosphorus (P) content during the research period. There was no significant difference in phosphrous (P) content during the study period.

Ammonia (NH3)
The decreasing ammonia on the 10 th day occurred on all treatment. It is also reported by Silviana et al. [15] that the initial ammonia concentration in the IMTA system is sharply increased but decreased drastically to stable on the 10 th day. The decreasing of ammonia concentration on this treatment revealed that G. verrucosa starts absorbing the ammonia in wastewater after the 10 th day through ammonification which is extracted for its growth. If the pH is 7 or less than that, it means that ammonia undergoes ionization. The pH during the experiment was approximately 7.42-8.83. When pH increased, a higher amount of ammonium will be absorbed by G. verrucosa. As a result, it will be decreased faster than nitrate and phosphate because of the ammonification process. G. verrucosa is proven to reduce ammonia concentration found in shrimp fishpond wastewater compared with control until at the end of the experiment. The plant weight on the 30 th day in treatment showed that there were significant differences with controls.

Nitrate (NO3)
Nitrate is the primary nitrogen form that exists in the natural water will quickly dissolve and is required for the algae growth. The increasing on the 10 th day on all treatments occurred as a result of physiological adaptation within the G. verrucosa to tolerate the high concentration of wastewater so that the nitrate absorption process was inhibited. Furthermore, wastewater on the 0 th day of treatment underwent a continuous nitrification process. The biofilter treatment of G. verrucosa was apparent on the 30 th day, while in the control treatment (without biofilter) showed increasing nitrate concentration. G. verrucosa started to absorb nitrate after the 20 th day.

Phosphate (PO4)
Phosphate is the phosphrous form that can be used by various plants such as water plants [9]. The increasing of phosphate concentration is caused by the evaporation of wastewater so that the phosphate concentration is higher than the previous. The pH condition during the research was ranged 7.42-8.83. The pH condition which higher than 7 will cause phosphate element in the form of H2PO4is reduced to H2PO4 2or in the form of PO4 3which furthermore is harder to absorb [6]. The phosphate concentration absorption done by G. verrucosa occurs on the 30 th day, which means that G. verrucosa as biofilter treatment is more prominent, while the control treatment without biofilter showed increasing. G. verrucosa biofilter becomes actively absorb the phosphate content after the 30 th day.
The decreasing phosphate content on the 30 th day occurred almost on all treatments except the control which experiences increase, indicating that G. verrucosa can absorb the phosphate content and make it as the nutrient for its growth. Phosphate compound has an interrupted cycle because it is reactive or easily bound to sediment but is hardly dissolve to the water [13]. G. verrucosa is considered effective to inhibit the increase of phosphate content found in the shrimp pond wastewater. The using of G. verrucosa resulted in a lower value of phosphate than control. The plant weight treatment did not affect the phosphate content on the wastewater.

G. verrucosa Biomass
Seaweed growth can be explained using a comparison between Carbon and Nitrogen (C/N ratio). The growth variously influenced by some environmental factors [4]. During the research, the biomass of G. verrucosa experienced increasing on the 10 th day. It was revealed that G. verrucosa can absorb the nutrient for its growth. However, the biomass tends to reduce on the 20 th and 30 th day since waste nutrient content decreased. It is also confirmed by Páez-Osuna et al. [12], that the less nutrient content in the water resulted in decreasing microalga biomass. Furthermore, the limited light factor in the laboratory has interrupted the photosynthesis process; hence, the biomass of G. verrucosa was decreased at the end of the treatment. Significant differences in the biomass of G. verrucosa showed on the 10 th , 20 th , 30 th days during the study period.

Absorption
Seaweed requires nitrogen and phosphorus elements for its growth. In general, the phosphorus element is absorbed in the form of orthophosphate, while nitrogen is in the form of nitrate, ammonium, and urea [1]. According to Nelson et al. [11], 500 grams of fresh seaweed Gracilaria sp. can reduce the total nutrient N concentration of 1.2 mg.L -1 to 0.4 mg.L -1 .
The result indicates that nitrogen (N) and phosphorus (P) content absorbed in the tip of G. verrucosa experienced increasing on all treatments to the end of the research, which caused by the ability of G. verrucosa in absorbing the nitrogen (N) and phosphorus (P) content to heal the tissue and grow the new one. The absorbed nitrogen (N) and phosphorus (P) content by G. verrucosa will cause its biomass addition, and increase the water quality.
The nitrogen (N) content in G. verrucosa tissue increased at the end of the research, from 0.14% to 0.19%. It is lower than the N and P absorption on G. lemaneiformis [17]. The absorption of nutrient waste by phytoremediation influenced by contact duration and phytoremediation density [10]. It becomes evident that G. verrucosa can absorb the content found in the waste for its growth. According to Abreu et al. [1], G. vermiculophylla absorption is decreased as the research duration and N accumulation on the tissue. Furthermore, N accumulation will be at the maximum value with the increase of N and NH4 + concentration. The phosphorus content (P) in G. verrucosa tissue increased at the end of the research, from 0.72% to 0.92%. It was observed that G. verrucosa can absorb N and P in wastewater for its growth that can be proved from the increasing of G. verrucosa fresh weight, and water quality.
The research concluded that G. verrucosa as biofilter can improve the water quality of wastewater by reducing the ammonia effectively on the 10 th day, nitrate on the 20 th day, and phosphate on the 30 th day. G. verrucosa absorbed inorganic nitrogen (N) content by 0.08%, and phosphorus (P) content by 0.35%. This research was funded by Universitas Gadjah Mada through RTA grant.