Comparison on Purification Effect of Two Macroalgae Species on Prawn Tailwater

Tingting ZHOU Jing HE Zhihua LIN Lin HE

Abstract ［Objectives］ This study was conducted to investigate the purification effects of two common large seaweeds on the tail water of prawn farming in greenhouses, and to determine the best culture density of seaweeds.

［Methods］Two large seaweed species, Gracilaria lichevoides and Ulva lactuca, were selected to set four culture densities of 0.5, 2, 4 g/L and a blank control group, respectively. The seaweeds were cultured in 100 L white polyethylene buckets, each of which contained 50 L of tail water from prawn culture.

［Results］ After 5 d, the nutrient removal rates of the two seaweeds were directly proportional to the density. There was no significant difference in NH4-N removal rate between G. lichevoides and U. lactuca (P>0.05) by two-way analysis of variance, and the NH4-N removal rate of the latter was higher. The removal rates of NO3-N, TN and TP by G. lichevoides were significantly higher than those by U. lactuca (P<0.05). The specific growth rates of seaweeds were negatively correlated with their culture densities. The specific growth rates of G. lichevoides were 5.73%, 1.654% and 0.48%, respectively, and those of U. lactuca were 2.01%, 1.187% and 0.138%, respectively, when the culture densities were 0.5, 2.0 and 4.0 g/L. Two-factor analysis of variance showed that the former was significantly higher than the latter, when the culture density of the two species of seaweeds was 0.5 g/L (P<0.05). The two-way analysis of variance showed that when the culture density of the two kinds of seaweeds was 0.5 g/L, the specific growth rate of G. lichevoides was significantly higher than that of U. lactuca (P<0.05). Based on the above research, the two macroalgae could reduce the nutrients in the wastewater to a large extent, but the culture density determined the scale and economic benefits of seaweed cultivation and further affected the normal growth, metabolism and quality of the seaweeds.

［Conclusions］This study provides some theoretical basis for large-scale seaweed farming and biological selection of in-situ ecological restoration of eutrophic seawater.

Key words Macroalga; Purification efficiency; Nutrient salt; Aquaculture tail water

Received: January 12, 2021  Accepted: March 11, 2021

Supported by Ningbo Citys 2015 Science and Technology Project for Enriching People: Optimization and Promotion of Prawn, Shellfish and Algae Ponds Integrated Aquaculture Technology (2015C10008); Ningbo Science and Technology Planning Project (2019C10039); Research and Development Project of Ecological and Efficient Clean Aquaculture of Mudflat Shellfish (2019C02054); China Shellfish Research System (CARS-49).

Tingting ZHOU (1991-), female, P. R. China, research assistant, devoted to research about ecological farming of aquatic organisms.

*Corresponding author. E-mail: hlwithyou@qq.com.

China is one of the more developed countries in marine aquaculture industry in the world, and its output also accounts for as much as 70% of the worlds total output, causing the pollution of the marine aquaculture industry to continue to increase. A large number of studies have proved that only about 20%-25% of the bait input in cage aquaculture is effectively used, and the rest is discharged into the water body in the form of residual bait and feces［1-2］. The study of Beveridge et al.［3］ showed that when trout digested 100 g of feed, and its fecal excretion was about 20-30 g in dry weight. Jia et al.［4］ found that during the culture of Litopenaeus vannamei from the larvae (0.02 g) to adult prawn (20 g), the accumulated N and P excretion amounts of a single cultured individual were 868.0 and 37.9 mg, respectively, and the accumulated manure N and P emissions were 218.3 and 190.8 mg, respectively. Research and investigations show that 300 000 t of finished prawn requires 390 000 t of feed, which means that 265 300 t of metabolites are discharged into the breeding environment［5］. With the rapid development of industrialization and high-density aquaculture, the eutrophication phenomenon of aquaculture tail water has become more and more serious, and its aquaculture quality and economic benefits have been greatly reduced, which has severely restricted the development of Chinas mariculture industry. Therefore, it is urgent to solve the pollution problem of mariculture tail water.

Gracilaria lichevoides and Ulva lactuca and other large seaweeds have a long life cycle, high productivity, and large biomass, and can absorb a large amount of nutrients such as N and P to achieve their own growth. Meanwhile, they increase the DO of the water body and play an important role in the purification of aquaculture tail water［2,6］. Qian et al.［7］ confirmed that Kappaphycus alvarezii has a certain repairing effect on Pinctada martensii culture areas. The characteristic of seaweed purification on water body is the symbiosis of plants and root zone microorganisms to produce a synergistic effect. Large seaweeds can remove N, P and suspended particles in the water through direct or indirect absorption, microbial transformation, physical adsorption and sedimentation and can decompose and absorb organic matter, and moreover, seaweeds can also absorb and enrich heavy metal molecules［8］. Luo et al.［9］ and Carmona［10］ have proved that large seaweeds can significantly remove nutrients from aquaculture tail water and have a good water purification effect. Li et al.［11］ and Gu［12］ studied the removal rate of inorganic N and P in aquaculture wastewater by U. lactuca and found that it had a good removal effect. Lyu［13］ found that large seaweeds had a certain ability to accumulate various heavy metals, and the accumulation was positively correlated with the concentration of heavy metals in seawater. The N and P in the water body are absorbed by seaweeds to synthesize its own structural components, and some heavy metals and organic substances that are toxic to alga are stored in plants or degraded in plants after detoxification［8］. At present, the concept of seaweed purification of eutrophic seawater has been recognized by scholars at home and abroad and has been vigorously promoted. However, the cultivation in different regions and of different seaweeds needs to be further refined and adapt to local conditions. Different seaweeds are different in the metabolic mechanism of organic matter absorbed due to their growth habit and contact area with water body. Therefore, choosing seaweeds with an appropriate density under different environments is the prerequisite to ensure the maximum comprehensive benefits. In this study, the nutrient removal rates of G. lichevoides and U. lactuca with different culture densities in the tail water were compared, hoping to provide a reference for the selection of seawater organisms for large-scale local seaweed cultivation and in-situ ecological restoration of eutrophication.

Materials and Methods

Experimental materials

This experiment was carried out in the prawn breeding workshop of Ningbo Ocean and Fishery Science and Technology Innovation Base, Zhejiang Province. The experiment period was 5 d. The experimental materials, G. lichevoides and U. lactuca were salvaged from the sea in Ningbo, Zhejiang Province by large ships. The experimental devices were 100 L white polyethylene plastic buckets, and the experimental water was the tail water of prawn farming in greenhouses.

Experimental methods

The experiment was a static experiment, and two large seaweeds, G. lichevoides and U. lactuca were selected. Four cultivation density groups were set for each type of alga, with 3 replicates in each group. The cultivation densities of the two algae were 0.5, 2 and 4 g/L, and a blank control group was also set. Before the experiment, the water on the surface of the seaweed was absorbed with absorbent paper, and then, its wet weight was measured. The seaweeds were cultured under DO 6.83-7.37 mg/L, salinity 25, water temperature 25.0-25.3 ℃, TN 4.01-4.45 mg/L, TP 0.56-0.65 mg/L, NH4-N 1.56-2.19 mg/L and NO3-N 0.35-0.76 mg/L with continuous aeration and natural light without water change during the experiment. Sampling was performed every day from 8: 30-9: 00.

Determination method: In this experiment, indexes such as NO3-N (nitrate nitrogen), NH4-N (ammonia nitrogen), TN (total nitrogen) and TP (total phosphorus) were selected for water quality analysis. Water sample collection method: One water sample (50 ml) was collected at each sampling point by immersing a sampling bottle under the water layer to take the middle part, shaking the bottle body to wash once, agitating the water body to remove the water layer foam, and quickly taking water. After sampling, the sampling bottle was put in a dark plastic bag and taken to the laboratory.

The water quality monitoring method referred to the marine monitoring specification［14］: NH4-N: DIN and ISO standard methods, TN: hydrazine sulfate reduction method, NO3-N: zinc cadmium reduction method, and TP［15］: TNTP combined digestion Mo-Sb-anti-spectrophotometry. During the experiment, the dissolved oxygen, salinity, and temperature in the water were measured using a dissolved oxygen meter (YSI).

Evaluation indexes: Nutrient removal rate (η)：η=(Co-CG)/Co×100%; specific growth rate of alga (%/d ): (SGR)=ln( Wt-W0 )/T×100%.

In the formulas, Co is the average concentration of water quality index before the experiment; CG is the average concentration of nutrient in the experimental group［16］; W0 is the wet weight of the seaweed at the beginning of the experiment; and Wt is the wet weight of the seaweed after t d.

Data analysis

The experimental results were expressed as the mean±standard deviation. The removal rates of different density groups and different species of seaweeds were analyzed and compared by two-way ANOVA in the Sigmaplot 13.0 software. The charts were drawn using Excel 2007, with P<0.01 indicating that the differences were extremely significant, P<0.05 indicating that the differences were significant, and P>0.05 indicating that the differences were not significant.

Results and Analysis

Nutrient removal rates of two kinds of seaweeds

The removal rates of NO3-N by different kinds of seaweeds: For the removal rate of NO3-N, the NO3-N removal rates of G. lichevoides in the 0.5 g, 2 and 4 g/L cultivation density groups and in the blank control group were 30.93%±3.57%, 36.89%±10.1%, 55.24%±10.7% and -27.13%±5.59%, respectively, and the NO3-N removal rates of U. lactuca with different culture densities were 21.27%±9.62%, 31.65%±6.525%, 35.78%±11.94%, and -25.33%±0.27%, respectively. After two-way analysis of variance, the removal rates of NO3-N in wastewater by two different seaweeds were significantly different (P<0.05). As can be seen from Fig. 1, the removal rates of NO3-N in the G. lichevoides groups were higher. In addition, the removal rates of NO3-N with different culture densities also had extremely significant differences. The different culture densities of the two kinds of seaweeds had significant differences with the blank control group. The removal rate of NO3-N was the highest at the culture density of 4 g/L, followed by 2 g/L, and the values were significantly higher than that of the blank control group (P<0.01).

The removal rates of TP by different kinds of seaweeds: There was a significant difference in the removal rate of TP in wastewater between the two kinds of seaweeds (P<0.05). G. lichevoides had a higher removal rate of TP. The removal rates of TP by G. lichevoides with culture densities of 0.5, 2 and 4 g/L and by the blank control group were 35.38%±6.67%, 47.94%±3.89%, 51.91%±4.57%, and 6.86%±1.06%, respectively. The removal rates of TP by U. lactuca at different cultivation densities were 25.57%±11%, 30.69%±0.34%, 37.92%±10.9%, and 7.83%±0.97%, respectively, and the two kinds of seaweeds showed significant differences between the cultivation densities of 2 and 4 g/L (P<0.05). The removal rate of G. lichevoides with a culture density of 4 g/L was the highest, which was significantly higher than that of the blank control group (P<0.01), as shown in Fig. 2.

The removal rates of TN by different kinds of seaweeds: In the two-way analysis of variance, there was no significant difference between the two seaweeds in the TN removal rate (P>0.05). It can be seen from Fig. 3 that the removal rate of TN by G. lichevoides was generally higher than that of U. lactuca, but there were significant differences in the removal rate of TN between the two seaweeds with different densities. The two kinds of seaweeds with a culture density of 4 g/L were both significantly higher than the respective blank control group and 0.5 g/L density group (P<0.05). The removal rates of TN by G. lichevoides with culture densities of 0.5, 2 and 4 g/L and  in the blank control group were 53.57%±8.98%, 66.68%±4.29%, 70.17%±6.1%, and -22.57%±7.8%, respectively, and the removal rates of TN by U. lactuca with different culture densities were 46.94%±4.54%, 53.27%±1.47%, 57.07%±8.83%, and -19.54%±6.17%, respectively.

The removal rates of NH4-N by different kinds of seaweeds: For the removal rate of NH4-N, the removal rates of NH4-N by G. lichevoides with the culture densities of 0.5, 2 and 4 g/L and in the blank control group were 88.96%±8.89%, 92.01%±0.38%, 93.45%±4.69 %, and -27.79%±6.15%, respectively, and the removal rates of NH4-N by U. lactuca with different culture densities were 90.86%±11.76%, 93.16%±9.93%, 95.75±1.77%, and -24.58%±1.97%, respectively, as shown in Fig. 4. The two-way analysis of variance showed no significant difference in the removal rate of NH4-N between the two seaweeds (P>0.05). U. lactuca of the two seaweeds had a higher removal rate of NH4-N, followed by G. lichevoides. However, seaweeds with different culture densities had significant differences in the removal rate of NH4-N, but the differences between seaweeds with culture densities of 2 and 4 g/L were not significant (P>0.05), and other groups had extremely significant differences (P<0.01).

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The same letter indicates a non-significant difference at the 0.05 probability level, and different letters indicate a significant difference.

Specific growth rates of seaweeds

After the experiment, the specific growth rates of the two seaweeds were determined. When the cultivation densities were 0.5, 2 and 4 g/L, the specific growth rates of G. lichevoides were 5.73%, 1.654%, and 0.48%, respectively, and the specific growth rates of U. lactuca were 2.01%, 1.187% and 0.138% respectively, as shown in Table 1. After two-way analysis of variance, the specific growth rate of G. lichevoides was significantly higher than that of U. lactuca (P<0.05) when the cultivation density of the two kinds of seaweed was 0.5 g/L, and there were significant differences in the specific growth rates of seaweeds between different densities. The specific growth rate of G. lichevoides in the 0.5 g/L group was significantly higher than those of other two groups (P<0.01), and the specific growth rate of U. lactuca in the 0.5 g/L group was significantly higher than that in the 4 g/L group (P<0.05).

Conclusions and Discussion

As one of the most promising bioremediators, large seaweeds can absorb dissolved CO2 and nutrients in water bodies through photosynthesis, and increase dissolved oxygen (DO) in water bodies to meet their own growth. In particular, the N content in water directly affects the survival and growth of aquaculture organisms, and the nitrogen in the aquaculture water mainly exists in the form of NH4-N, NO3-N and NO2-N. In this study, the removal rates of NH4-N by G. lichevoides and U. lactuca were significantly higher than the removal rates of NO3-N and TP, and the maximums reached 93.54%±4.69% and 95.75%±1.77%. Many scholars have also found similar findings. Skriptsova et al.［17］ found in their research laboratory simulation system that the removal rates of NH4-N by U. lactuca and G. lichevoides were in the range of 90%-99%. Mc Glathery［18］ also found that NH4-N could be absorbed by macroalgae for their own growth, and it was preferentially absorbed compared with nitrate and nitrite. Feng［19］ believed that when NH4-N and NO3-N coexisted under the same conditions, seaweeds would preferentially absorb NH4-N, and when its concentration was lower than 0.01 mg/L, seaweeds would start to absorb a large amount of NO3-N. Some scholars［20-21］ also believed that algae first absorbed NH4-N in the environment because it could be directly absorbed by the algae and synthesize N-containing organic substances such as amino acids through transamination. In this study, it was found that the NH4-N removal rate of U. lactuca was higher than that of G. lichevoides, which might be related to the larger surface area of U. lactuca. The study of Pederson et al.［22］ showed that the degree of coupling between the growth of large seaweeds and the absorption of N was related to the surface area-to-volume ratio of seaweeds. However, for the removal rates of TP and NO3-N, the values of G. lichevoides were significantly higher than those of U. lactuca (P<0.05). There are many factors that affect the absorption rate of P, and the concentrations of N and P in the water, the ratio of nitrogen to phosphorus, and the concentration of phosphate in the body of G. lichevoides all affect its absorption of phosphorus in water［23］. In addition, the culture density of the two seaweeds was positively correlated with their nutrient removal rates. Both seaweeds had the highest nutrient removal rates at 4 g/L, but the removal rates were not much different from those at 2 g/L. Considering that the density of seaweeds in actual production should not be too high, therefore, it is more suitable to cultivate seaweeds with a density of 2 g/L. For the removal rate of NH4-N, the value of U. lactuca was higher than that of G. lichevoides, but there was no significant difference between the two. The removal rates of other three nutrients by G. lichevoides were all higher than those by U. lactuca.

Both seaweeds grew during the experiment, and the specific growth rates of the two seaweeds were negatively correlated with their cultivation densities, but the specific growth rate of G. lichevoides was significantly higher than that of U. lactuca (P<0.01). Troell et al.［24］ conducted research on G. lichevoides raised in open salmon cages and showed that it had certain removal effects on inorganic nitrogen and phosphorus in the cultured seawater and the growth rate of G. lichevoides was improved. The results of this study showed that both seaweeds had high removal rates of nutrients in the water body and had the function of bioremediation. However, in terms of purification ability or specific growth rate, G. lichevoides was better than U. lactuca. In addition, G. lichevoides is a kind of seaweed that can reproduce vegetatively, and after one time of seedling input, it can be harvested regularly as the alga can continuously grow and reproduce by retaining part of it［25］, so it is suitable for cultivation in the waters of Zhejiang Province and is an ideal species for bioremediation.

References

［1］ LI Y, WANG L, JIANG LY, et al. Aquaculture nutrition, eco-fitness and environmental protection［J］. Marine Sciences, 2004, 28(3): 76-78. (in Chinese)

［2］ LIN ZX, RU SG, YANG YF. Prospect for bioremediation of large-sized seaweed cultivation in eutrophic bays［J］. Transactions of Oceanology and Limnology, 2006(4): 128-134. (in Chinese)

［3］ BEVERIDGE MCM, et al. Aquaculture and water quality, advances in world aquaculture［C］ Louisi-ana World Aquaculture Society.1991(3):456-467.

［4］ JIA XP, LI ZJ, JIANG SG, et al. Application of microbial engineering technology in large-scale prawn farming［R］. Guangzhou: South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, 2003: 119-141. (in Chinese)

［5］ LI ZJ, CHEN YQ, YANG YY, et al. Prevention and control countermeasures on environmental pollution from prawn culture in Guangdong ［J］. Guangdong Agricultural Sciences, 2006(6): 68-71. (in Chinese)

［6］ YUE WZ, HUANG XP, HUANG LM, et al. Preliminary study on purification of mariculture water by macroscopic algae［J］. Marine Environmental Science, 2004, 23(1): 13-15. (in Chinese)

［7］ QIAN PY, WU CY, WU M, et al. Integrated cultivation of the red alga Kappaphycus alvarezii, and the pearl oyster Pinctada martensi［J］. Aquaculture, 1996, 147(1-2):21-35.

［8］ TU XC, CAI MZ, SUN JG. Purification and mechanism of aquatic macrophytes in polluted water［J］. Journal of Anhui Agricultural Sciences, 2006, 34(12): 2843-2844. (in Chinese)

［9］ LUO YS, LI ZJ, WEN GL, et al. Preliminary studies on density changes of Gracilarla tenuistipitata var. liui used for the purification of prawn effluent［J］. South China Fisheries Science, 2006, 2(5): 7-11. (in Chinese)

［10］ CARMONA R, KRAEMER GP, YARISH C. Exploring Northeast American and Asian species of Porphyra, for use in an integrated finfish-algal aquaculture system［J］. Aquaculture, 2006, 252(1):54-65.

［11］ LI XC. Static purification of Ulva pertusa Kjellman for abalone effluents［J］. Transactions of the Chinese Society of Agricultural Engineering, 1998, 14(1): 173-176. (in Chinese)

［12］ GU H, XU J, ZHANG XM, et al. Absorption of Ulva Pertusa to nutrient salt in piscatorial wastewater［J］.  Environmental Science & Technology, 2007, 30(7): 85-87. (in Chinese)

［13］ LYU LY. Study on bioremediation ability of several seaweeds on heavy metal contamination in seawater［D］. Qingdao: Ocean University of China, 2013. (in Chinese)

［14］ Standardization Administration of the Peoples Republic of China. GB 17378.4-2007 Marine monitoring norms part 4: Seawater analysis ［S］. Beijing: Standards Press of China, 2007.

［15］ XING DL, HUO TB, WU HM, et al. Simultaneous digestion for determination of total phosphorus and total nitrogen in sea water ［J］Journal of Dalian Fisheries University, 2006, 21(3): 219-225.

［16］ MA XN, LI M, SUN GX, et al. Absorption of Ulva Pertusa to nutrient salt in piscatorial wastewater［J］. Marine Sciences, 2016, 40(1): 32-39. (in Chinese)

［17］ SKRIPTSOVA AV, MIROSHNIKOVA NV. Laboratory experiment to determine the potential of two macroalgae from the Russian Far-East as biofilters for integrated multi-trophic aquaculture (IMTA)［J］. Bioresource Technology, 2011, 102(3): 3149.

［18］ MCGLATHERY KJ, PEDERSEN MF, BORUM J. Changes in intracellular nitrogen pools and feedback controls on nitrogen uptake in Chaetomorpha linum, (chlorophyta) 1［J］. Journal of Phycology, 1996, 32(3): 393-401.

［19］ FENG MX. Study on bioremediation of eutrophic seawater by Gracilaria lichevoides［D］. Fuzhou: Fujian Normal University, 2011. (in Chinese)

［20］ NAVARRO-ANGULO L, ROBLEDO D. Effects of nitrogen source, N: P ratio and N-pulse concentration and frequency on the growth of Gracilaria cornea (Gracilariales, Rhodophyta) in culture［J］. Hydrobiologia, 1999, 398-399(3): 315-320.

［21］ DEBOER JA, GUIGLI HJ, ISRAEL TL, et al. Nutritional studies of two red algae. I. Growth rate as a function of nitrogen source and concentration［J］. J. Phycol., 1978(14): 261-266.

［22］ PEDERSEN MF, PALING EI, WALKER DI. Nitrogen uptake and allocation in the seagrass Amphibolis antarctica［J］. Aquatic Botany, 1997, 56(2): 105-117.

［23］ Parsons TR, et al. Biological oceanographic processes ［J］. Quarterly Review of Parsons T R, 1977, 44(1):116-127.

［24］ TROELL M, HALLING C, NILSSON A, et al. Integrated marine cultivation of Gracilaria chilensis (Gracilariales, Rhodophyta) and salmon cages for reduced environmental impact and increased economic output. ［J］. Aquaculture, 1997, 156(1-2): 45-61.

［25］ TANG KX, YUAN DX, LIN SB, et al. Depression and affect of red tide on main water quality index by Gracilaria tenuistipitata［J］. Marine Environmental Science, 2003, 22(2): 24-27. (in Chinese)