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MINISTRY OF EDUCATION AND TRAINING CAN THO UNIVERSITY SUMMARY of THE PhD THESIS Major: Soil and water environment Code: 62440303 BUI THI MAI PHUNG STUDY ON APPLYING SEDIMENT AND MICROALGAE TO IMPROVE THE INTENSIVE RICE SOIL ENVIRONMENT OF THE FULL-DYKE SYSTEMS IN AN GIANG PROVINCE Can Tho, 2020 THIS STUDY WAS ACHIEVED AT CAN THO UNIVERSITY Scientific Supervisor: Assoc Prof Dr Nguyen Huu Chiem This dissertation was defended at the University Examination Council At: …………………………………… At…hour , on date…month….year… Reviewer 1:…………………………… Reviewer 2:…………………………… The dissertation is available at: Learning Resource Center of Can Tho University National Library of Viet Nam LIST OF PUBLICATIONS [1] Bui Thi Mai Phung, Huynh Cong Khanh, Pham Van Toan and Nguyen Huu Chiem, 2017 Study on the quantity and nutrients content of sediment in the full-dyke and semidyke systems in An Giang province Journal of Science, Can Tho University (ISSN 18592333), thematic issues: Environment and Climate change, 1: 146-152 [2] Bui Thi Mai Phung and Nguyen Huu Chiem, 2018 Study on the role of sediment helps to improve rice yield in net houses Journal of soil science (ISSN 2525-2216), 53: 25-30 [3] Bui Thi Mai Phung, Vo Dan Thanh and Nguyen Huu Chiem, 2019 Diversity of benthic microalgal species in intensive rice cultivation in Cho Moi district, An Giang province, Vietnam Journal of Science, Can Tho University (ISSN 1859-2333), thematic issues: Environment, 55: 53-67 CHAPTER 1: INTRODUCTION 1.1 Introduction An Giang is the upstream province of the Mekong River when it enters Vietnam and is the leading rice-producing province in the Mekong Delta Cho Moi was the first district to carry out the full-dyke systems in 1995 in order to produce third-crop rice so increasing rice production per unit area The intensive rice cultivation field has caused some disadvantages, such as soil without flood discharge, so the toxin has stagnated in the soil (Tran Nhu Hoi, 2005), and pesticides content increases, copper ions content derived from pesticides are easily washed from rice fields into waterways of canals (Tran Thanh et al., 2014) There were a loss of nutrients from sediment (Truong Thi Nga, 1999; Nguyen Van Nha, 2005; Bui Dat Tram, 2006; Duong Minh Vien et al., 2010; To Van Truong, 2014), from microalgae and Cyanobacteria because floodwaters can contribute an increase of the Cyanobacteria population by 4.5 times during the flood season (Begum et al., 1988) Chlorophyta, Bacillariophyta, Euglenophyta and Cyanobacteria often grow in the surface water or topsoil in the rice fields Therefore, after each period of fertilization for rice plants, especially applying phosphorus fertilizer, the microalgae grew very fast, so their algal biomass in the fields increased significantly When they die, they can be an available source of organic matter for the soil Moreover, Cyanobacteria have the ability to fix nitrogen from the atmosphere to the soil by heterocysts (Duong Duc Tien, 1996; Nguyen Van Tuyen, 2003; Vu Quang Manh, 2004) and increase the water holding capacity of the soil by 40% Thus, the combination of hundreds of species of microalgae and other microorganisms contributed to keeping the equilibrium and stability of wet rice ecosystems (Nguyen Van Tuyen, 2000) The research result of Nguyen Huu Chiem et al in 2017 in An Giang province showed that the content of organic matter, total nitrogen and phosphorus of the soil inside of the full-dyke systems were significantly different than that outside of the full-dyke (p < 0.05) although the full-dyke duration was nearly twenty years Especially, amount of nitrogen and phosphorus fertilizers applied inside of the full-dyke is significantly higher than outside of the full-dyke The question is why the content of organic matter, total nitrogen and phosphorus in the soil is high, but the rice yield does not increase? However, additional nutrients sources for the soil such as rice straw, sediment or microalgae have not been published, especially some studies on biomass and nutrient of microalgae in intensive rice cultivation fields? Therefore, this study was conducted to determine their function in improving the soil environment 1.2 Objectives of study 1.2.1 General objective Study on the ability to supply total weight of deposited sediment, biomass and nutrients of microalgae and deposited sediment for the rice soil inside and outside of the full-dyke systems 1.2.2 Specific objective Determination of total weight and nutrient content of deposited sediment have inside and outside of the full-dyke systems Determination of dominant phyla, biomass, and total NPK content of microalgae provided with the intensive rice cultivation fields Determining contribution rate of deposited sediment and microalgae for the soil annually and evaluating their function improve the soil 1.3 The contents of the study Determination of total weight and physical and chemical properties of sediment inside and outside of the full-dyke in An Giang province from 2013 to 2015 Determining species diversity, density and estimating the total biomass and nutrients of planktonic and benthic microalgae provided with intensive rice cultivation fields annually Estimating the contribution rate of deposited sediment and microalgae for the soil and evaluating their function improve the soil 1.4 New findings of the thesis and the practical significance of the study The thesis has proven the function of deposited sediment from the river as an available source of nutrients for the soil, so the soil can receive an amount of nutrients equal to 3.85%, 3.95% and 40.3% of the chemical fertilizers (N, P2O5 and K2O, respectively) The content of organic carbon and total phosphorus in the soil after harvest of the additional treatments with sediment increased from 1.29 to 1.59 times compared with in the soil before planting A single linear regression equation has been established between the deposited sediment weight and the time of flooding The thesis determined four hundreds and forty-five taxa of planktonic and benthic microalgae in the rice fields, so they can modify the data set of species composition in the Mekong Delta In particular, six taxa of Cyanobacteria have the ability to fix nitrogen in the soil by heterocysts, in which Anabaena Oscillarioides appeared with high density at the beginning of the tilling stage in the Summer-Autumn rice of 2017 The biomass of planktonic and benthic microalgae was estimated about 1.08 tons fresh biomass per per year, so amount of nutrients of microalgae can provide with the soil equal to 0.465 kgN/ha, 0.197 kgP/ha and 1.26 kgK/ha but not estimated nitrogen content fixed by Cyanobacteria Thus, the actual content of nitrogen of the microalgae can provide with the soil a lot The thesis has also established an image collection of microalgae living in the rice fields The microalgae catalogs were arranged in an easy and most convenient way for reference The research results of the thesis are a source of scientific data for managers in relation to environment and agriculture The study helps planners in arranging rice production patterns combined with flood control and changing new techniques to allow microalgae growth and fix nitrogen for the soil Besides, the thesis is also a valuable data source for further study, research and teaching in Institutes and Universities CHAPTER 2: LITERATURE REVIEWS 2.1 The concept of sediment and its function improve the soil In 1998, in the area of My Duc commune, Chau Phu district, An Giang province, the weight of deposited sediment was about 100 tons/ha (Truong Thi Nga, 1999), in 2000 in the north of Vam Nao I area was 35 tons/ha and the north of Vam Nao II area was 80 tons/ha (Duong Van Nha, 2003) The flood discharge into the soil contributes to the retention of sediment on topsoil and the weight of deposited sediment was high from July to August (Le Xuan Thuyen et al., 2000) There have been many studies in An Giang province for many years They proved that the depoised sediment was a source of available nutrients to improve the soil and plants The deposited sediment could help to maintain the fertilization of the soil (Tran Thuong Tuan et al., 1999), and contribute to slowing the depletion of nutrients of the soil that the soils receive the deposited sediment annually (Duong Minh Vien et al., 2010) Besides, the sediment itself with a light texture played a role in improving the physical properties of the soil Due to intensive rice cultivation, the rice straw cannot quickly decompose and mineralize to provide organic humus and nutrients for the soil, so there was a possibility that the soil would harden (Tran Thuong Tuan et al., 1999) Because of the lack of deposited sediment for the soil inside of the full-dyke systems, the weight of chemical fertilizers applied to rice plants increased to compared with before establishing the full-dyke (Tran Nhu Hoi, 2005; Duong Van Nha, 2005; Huynh Dao Nguyen and Vo Thi Guong, 2010a; Tran Anh Thu et al., 2013) Flood discharge into the rice fields can help retain sediment on the topsoil (Le Xuan Thuyen et al., 2000), contribute to supply nutrients for the soil, so it could be utilized to grow a rice crop (one crop per year) without fertilizer with rice yield about three tons of rice per hectare Because nutrients of sediment have provided enough for rice-plants in one crop (Nguyen Bao Ve, 2011) Thereby, it showed that the weight and nutrients of deposited sediment are valuable for the soil 2.2 The concept of microalgae and its function to improve the soil Microalgae are all microscopic sized algae, which are the lowest plants They belong to the group of photosynthetic autotrophic, living freely in the surface water (Hoang Thi San, 2009) that is called planktonic microalgae; and the microalgae live on the bottom of soil, stick on living things or into boats that is called benthic microalgae (Nguyen Nghia Thin and Dang Thi Sy, 1998) It is based on specific photosynthetic pigments or the shape of each algae phylum that identify them, such as Chlorophyta with green color, Bacillariophyta with yellow, Cyanobacteria with blue-green, or Euglenophyta with vein patterns There are always four phyla such as Chlorophyta, Bacillariophyta, Euglenophyta and Cyanobacteria (Figure 2.1 and Figure 2.2) in the rice fields in Vietnam and the world (a) (b) (c) (d) Figure 2.1: Some species belonging to Bacillariophyta (Boyer et al., 1916) and Cyanobacteria (Bellinger and Sigee, 2015) (a): Surirella elegans Ehr, (b): Stephanopyxis corona (Ehr.) Grun, (c): Microcystis, (d): Anabaena flos-aquae Some Cyanobacteria genera fixed nitrogen in the rice fields such as Anabaena, Anabaenopsis, Aphanothece, Aphanizomenon, Aulosira, Calothrix, Cylindrospermum, Nostoc, Nudolaria, Lyngbya, Tolypothrix, Rivularia, etc (a) (b) (c) (d) Figure 2.2: Some species belonging to Chlorophyta and Euglenophyta (Nguyen Lan Dung and Nguyen Hoai Ha, 2006) (a): Phacotus lenticularis, (b): Hydrodictyon reticulatum, (c): Pediastrum boryanum, (d) Phacus tortus Algae in water bodies in general and in rice fields in particular was affected by light, temperature, humidity, pH, concentration of nitrogen and phosphorus, the rice leaf canopy and grazers The grazers include Cladocera, Copepoda, Ostracoda, etc The algae can provide nitrogen for the soil by fixing nitrogen; return the nutrients when they die; and provide dissolved oxygen, so they help the balance of the ecosystem of rice fields Several factors influenced capable of fixing nitrogen of Cyanobacteria such as physical, chemical and biological factors; and farming techniques Some species of Cyanobacteria with heterocysts have the ability to fix nitrogen from the atmosphere into the soil The following nitrogen fixation reactions occur: (2.1) (2.2) The nitrogen content was fixed in natural soil by Cyanobacteria was 14 kgN/ha (De and Mandal, 1956 cited by FAO of the United Nations, 1981) During the appropriate season, the fixed nitrogen content was from 20 to 30 kgN/ha (Kaushik, 1994; Dang Dinh Kim and Dang Hoang Phuoc Hien, 1999) or phosphorus fertilizer was added into the soil that the nitrogen content of the soil increased from 18 to 69 kgN/ha The fixed nitrogen content of the soil increased from to 8% total nitrogen after 30 days of culturing Cyanobacteria (Nguyen Xuan Thanh et al., 2009) or increased 31.6% compared with the control treatment (Yagya and Shreeti, 2012) Depending on the kinds of Cyanobacteria, the kinds of amino acids are released into the soil differently For example, Calothrix brevissima released alanine, aspartic and glutamic, while Nostoc muscorum released glutamic and ammonia into the soil (Nguyen Xuan Hien et al., 1975) The spraying of Cyanobacteria strains on rice plants also helped to improve the soil The pH value, humus, total nitrogen and available nitrogen of the soil increased 10.7%, 4.83%, 5.56% and 8.92% compared with the control treatment, respectively (Nguyen Dinh San, 2015) After three years of adding Cyanobacteria on the fields, the content of organic carbon of the soil increased 68.7% compared with the control treatment (Singh, 1961 cited by Roger and Kulasooriya, 1980) Some Cyanobacteria genera can help to increase the rice yields There are Tolypothrix (in Japan), Anabaena (in India, Nepal, Iran, and Vietnam), Calothrix (in Vietnam), Nostoc (in Bangladesh and Vietnam), Aphanothece and Gloeotrichia genera (in Vietnam) (Nguyen Xuan Hien et al., 1975; Duong Duc Tien, 1990; Nguyen Dinh San and Le Thanh Tung, 2007; Begum et al., 2011; Alam et al., 2014) If farmers utilized them effectively, the amount of chemical fertilizers applied to the rice could be reduced from 15 to 30% of the total amount of the chemical fertilizers (Kaushik, 1994, Dang Dinh Kim and Dang Hoang Phuoc Hien, 1999) CHAPTER 3: METHODOLOGY 3.1 Research place and time The research was carried out in four districts of An Giang province and An Giang University (AGU) from 2013 to 2018 The experimental samples were analyzed at the Intensive Laboratory, Department of Soil Chemistry – Faculty of Agriculture and Applied Biology and Environmental Quality – Faculty of Environment and Natural Resources in Can Tho University (CTU); and Environment and Agriculture Laboratories in An Giang University (AGU) 3.2 Methodology 3.2.1 Methods of experimental arrangement and field survey 3.2.1.1 Content 1: Determination of total weight of deposited sediment and its function in improving the soil and the weight of filled rice grains a Determination of total weight and physical and chemical properties of deposited sediment inside and outside of the full-dyke systems in An Giang province from 2013 to 2015 (Content 1.1) Study place and time: The content was carried out from August in 2013 to May in 2015 and in four districts such as Chau Phu, Phu Tan, Thoai Son and Cho Moi district that have a relatively large area of full-dyke in An Giang province The time for installing sediment traps inside and outside of the full-dyke were the same time Sediment traps were set in August and collected in December They were made of nylon with one m2 (1 × m) They placed on topsoil in the rice field, and anchored with four stakes at four corners during the flood season (Figure 3.1) (a) (b) Figure 3.1: Sediment traps was set inside (a) and outside (b) of the full-dyke in Thoai Son district in 2013 in An Giang province Twenty houndreds ninety-six samples of deposited sediment were collected from 2013 to 2015, of which 148 samples were collected inside and 148 samples outside of the full-dyke The collected sampling positions were located by using a global positioning device (GPS), and the coordinates were transferred to a Google map Some parameters were identified such as total weight, texture, CEC, the content of organic matter and total NPK of deposited sediment, and the time of flooding b Study on the function of sediment in improving the soil and the weight of filled rice grains in the pot experiment (Content 1.2) The pot experiment was carried out in two rice crops such as in the Winter-Spring rice of 2013-2014 (pre-test) and the Winter-Spring rice of 2014-2015 in the net house of An Giang University The first experiment was arranged by a completely randomized design with one factor (fertilizer), three replications with nice treatments The weight of additional sediment increased gradually from 0.4, 0.8, 1.2, 1.6, 2.0 to 2.4 kg and that of soil decreased from 5.0 to 2.6 kg to obtain a total weight of 5.0 kg in each pot The result of the first experiment showed that no significant difference about the weight of filled grains between two treatments (the weight of additional sediment was 0.4 kg/pot and 0.8 kg/pot) (p > 0.05) Therefore, the next experiment was carried out The pot experiment was arranged by a completely randomized design with two factors (fertilizer and sediment), three replications with eight treatments Each replication was a pot, transplanting three pieces of rice The weight of additional sediment increased from 0.4, 0.8, 1.2 to 2.4 kg and that of soil decreased from 5.0 to 2.6 kg to obtain a total weight of 5.0 kg in each pot (Table 3.1) Table 3.1: Experimental treatments with sediment in the pot experiment in the WinterSpring rice of 2014-2015 Treatment Describe Fertilizer Treatment (NT1) kg rice soil 0% Treatment (NT2) kg rice soil 100% Treatment (NT3) 0.4 kg sediment + 4.6 kg rice soil 0% Treatment (NT4) 0.4 kg sediment + 4.6 kg rice soil 100% Treatment (NT5) 1.2 kg sediment + 3.8 kg rice soil 0% Treatment (NT6) 1.2 kg sediment + 3.8 kg rice soil 100% Treatment (NT7) 2.4 kg sediment + 2.6 kg rice soil 0% Treatment (NT8) 2.4 kg sediment + 2,6 kg rice soil 100% The experimental soil and sediment have a texture of clay loam, the content of nutrients was range in the average to high levels, and the conductivity (EC) of soil and sediment were less than mS/cm (Table 3.2) In general, they not have the limiting factors in rice cultivation, but the nutrients of the sediment were higher than that of the soil Table 3.2: Physical and chemical properties of the soil and sediment before planting in the Winter-Spring rice of 2014-2015 Parameter Unit % sand Rice soil Sediment % 1.40 0.769 Texture (%) % loam % 55.2 41.4 43.4 57.9 4.52 6.70 % clay % Water pH EC mS/cm 0.22 1.12 Organic C % 4.32 6.03 CEC meq/100 g 10.7 13.3 K+ meq/100 g 1.50 2.25 meq/100 g 20.4 37.3 2+ Ca Parameter Unit Rice soil Sediment Mg2+ meq/100 g 1.72 2.21 Na+ meq/100 g 0.60 0.45 Total nitrogen %N 0.20 0.44 Total phosphorus %P2O5 0.04 0.06 Total Potassium %K2O 1.99 2.04 Ammonium mg/kg 18.9 39.9 Available phosphorus mg/kg 5.42 15.8 After twenty days of sowing rice seeds OM 6976, choosing seedlings of uniform size and transplanting three seedlings into a pot 120 kgN - 60 kg P2O5 - 30 kg K2O was applied for every hectare of rice soil Fertilizers were divided into three stages such as: the first stage (20 days after sowing), the second stage (45 days after sowing) and the third stage (65 days after sowing) In all three stages, 0.083 g N per pot was applied, especially the first stage applied 0.150 gP2O5/pot; the second and the third stage applied 0.0375 gK2O/pot During the process of planting, weeding, catching worms, watering and spraying pesticides to prevent and kill pests and diseases throughout the experiment Measurement parameters such as the weight (g/pot) and percentage of filled grains (%) at 14% standard moisture when harvesting, and the content of organic carbon (%) and total NPK (%) in the soil after cultivating c Evaluating the function of deposited sediment in improving the soil (Content 1.3) Statistics comparing the chemistry data of soil inside and outside of the full-dyke in An Giang province from 2013 to 2015 from Nguyen Huu Chiem et al in 2016 The data was applied to evaluate the function of sediment 3.2.1.2 Content 2: Evaluation of species diversity and the ability to provide biomass and nutrition of microalgae for the soil environment d Evaluation of species diversity and the ability to provide biomass and nutrition of planktonic and benthic microalgae in the rice fields in Cho Moi district (Content 2.1) The content was carried out in all three rice crops such as Autumn-Winter rice of 2016 (from August to December in 2016), Winter-Spring rice of 2016-2017 (from December in 2016 to March in 2017), and Summer-Autumn rice of 2017 (from April to August in 2017) Three rice fields were selected with each rice field area being 1,000 m2 with the same varieties, sowing density, fertilizers, pesticides and water management Before sowing, the rice straw in the rice fields was not buried, but only ploughed the stumps Method of setting artificial substrates: refined brick (size 170×70×35 mm) was cut into four smaller pieces (size 85×35×35 mm) made of substrates for benthic microalgae in the rice fields Before sowing rice, 20 bricks were placed in each rice field at some positions as shown in Figure 3.2; the substrates were placed on topsoil so that the thickness of the bricks on the ground was 10 mm leading to water surface area exposed to sunlight decrease, so the intensity of light on the field is low Therefore, the photosynthesis process of microalgae is limited resulting in reduced carbon dioxide consumption, so the pH values and dissolved oxygen concentration reduce Besides, ammonium, nitrate and phosphate concentrations tended to decrease gradually from two to six days after fertilizing because of absorption of nutrients by the rice plant and microalgae Figure 4.8: Phosphate concentration in rice fields in three rice crops Figure 4.9: Dissolved oxygen concentration in rice fields in three rice crops * Comparison of the quality of water in the rice fields in three rice crops Table 4.12 shows that there were significant differences in temperature, pH value, ammonium and nitrate concentration of water in the field between three rice crops (p < 0.05) but no significant differences in phosphate and dissolved oxygen concentration of the water in the field (p > 0.05) Because dissolved oxygen concentration of the water can change seasons, weather, day, night, and depth The solubility of oxygen in water decreases as water temperature and salinity increase Table 4.12: The comparing statistics of physical and chemical parameters of water in the field in three rice crops Rice crop Water to o ( C) Water pH Ammonium Nitrate Phosphate DO -mg/L Autumn-Winter 29.5±2.29b 6.62±0.449a 0.484±0.539b 0.482±0.457a 0.089±0.076 4.47±3.20 Winter-Spring 27.5±1.91c 6.80±0.453a 0.881±0.877a 0.284±0.184b 0.097±0.117 5.30±2.61 a b a a 0.105±0.074 4.61±3.81 ** ns 0.67ns Summer-Autumn F value 31.3±1.82 32.2 ** 6.23±0.516 13.8 0.976±0.713 ** 4.49 * 0.643±0.532 6.62 0.3 Note: n = 36 samples, in the same column, the same followed digits are not significantly different at the significance level of 0.05, *: significant difference of 0.05 levels, **: statistically significant difference at 0.01 levels according to the Duncan test Comparison of the quality of water in the field with QCVN 08-MT: 2015/BTNMT, applying B1 column (water is used for irrigation purposes), the results show that the pH value (5.72-7.56) and the nitrate concentration (0.057-1.40 mg/L) of water in the field were below the allowed threshold, so they were evaluated to achieve the standard Therefore, the pH of water in the field has not yet affected the pH property of soil, but applying more chemical fertilizers such as fertilizers NH4NO3 and (NH4)2SO4 fertilizers for a long time resulting in the soil being acidic The research of Hanoi General University showed that applying NH4NO3 fertilizer to plants which was grown in the sediment soil in the Red River after four years, the pH values of the soil reduced from 6.9 26 to 5.4 Applying (NH4)2SO4 fertilizer to non-acid soil, NH4+ ions are absorbed into the soil colloid and push Ca2+ ions out of the soil colloid, causing the soil to lose Ca2+ ions, making the soil gradually acidic for a long time (Le Van Khoa et al., 2010) Beside that, applying inorganic nitrogen fertilizers continuously, it affected the acidification of the cultivated layer The nitrate concentration of 13.8% of wells that were surveyed in the delta was mg/L (Tran Van Chinh et al., 2006) However, the concentration of ammonium, phosphate and dissolved oxygen of water in the field at some time exceeded the allowed threshold of the standard Dissolved oxygen concentration was lower than mg/L; it indicates that the concentration of organic matter of water in the field was very high If water in the field is discharged continuously into the local canal ínide the fulldyke systems, the nitrogen and phosphorus of water are the two components that can cause eutrophication 4.2.2 Presence of microalgal phyla and density of microalgae in the rice fields in three rice crops 4.2.2.1 Species diversity and density of planktonic microalgae in three rice crops a Species diversity of planktonic microalgae in the rice fields Table 4.13 shows that there were 407 planktonic microalgae taxa belonging to 110 genera, 39 families, 16 orders, and four phyla (Chlorophyta, Bacillariophyta, Euglenophyta and Cyanobacteria) in the intensive rice cultivation fields, in which Chlorophyta predominated, followed by Bacillariophyta, Euglenophyta and Cyanobacteria Importantly, there were five Cyanobacteria taxa have ability fix-nitrogen such as Anabaena affinis Lemm, Anabaena circinalis, Anabaena oscillarioides (Figure 4.10), Anabaenopsis elenkinii and Aphanizomenon flos-aquae (Nguyen Van Tuyen, 2003) Figure 4.10: Anabaena oscillarioides with heterocycts appeared in the intensive rice cultivation fields in Summer-Autumn rice of 2017, Cho Moi district, An Giang province Table 4.13: The structure of species composition of planktonic microalgae in the fields in three rice crops Phylum Order Family Genus Species Percentage of species Bacillariophyta 25 101 24.8 Chlorophyta 21 52 163 40.1 Euglenophyta 1 90 22.1 Cyanobacteria 18 53 13.0 16 39 100 407 100 Total There was a great variation in the number of algae species in three rice crops, especially the number of species in the Summer-Autumn rice (276 species) was higher than the Autumn-Winter rice (88 species) Because the environmental conditions in the two crops are quite different such as rainfall, cloudy sky and temperature, and spraying of plant protection chemicals to prevent pests and diseases on the rice This activity 27 influenced microalgae because the activities of pesticides have the ability to kill algae These are copper oxychloride, butachlor, propanil, abamectin, permethrin, pymetrozine and difenoconazole In general, the total number of algae species was determined in this study is more than in the previous studies in the Mekong Delta and Ha Noi For example, the research of Nguyen Huu Chiem et al (1999), Duong Tri Dung et al (2002), and Ngo Ngoc Hung (2009) in the Mekong Delta and Ngo Thanh Trung et al (2008) in Ha Noi This research conducted surveys according to each development stage of rice plants and rice crops, while the previous studies only surveyed one crop or a short time b Density of planktonic microalgae in the rice fields Table 4.14 shows that the total density of planktonic microalgae ranged from 11,063 to 25,837 individuals per liter The total density of algae between the three rice crops was not significantly different (p > 0.05) because the density of Euglenophyta in the Summer-Autumn rice was lower than in the Winter-Spring rice, but the density of Cyanobacteria in the Summer-Autumn rice was higher than in the Autumn-Winter rice (p < 0.05) Meanwhile, the density of Bacillariopyta and Chlorophyta in all three crops was not significantly different (p > 0.05) Table 4.14: Density of planktonic microalgae (Individual/L) appeared in the rice fields in three rice crops Rice crop n Bacillariophyta Chlorophyta Euglenophyta Cyanobacteria Total Autumn-Winter 36 7,282 2,564 6,689ab 1,601b 18,094 Winter-Spring 36 16,822 1,300 7,413a 301b 25,837 Summer-Autumn 36 2,781 1,940 1,283b 5,059a 11,063 F value 1.95ns 0.533ns 2.68** 12.0** 1.42ns Note: In the same column, the same followed digits are not significantly different at the significance level of 0.05, ns: no significant difference at 0.05 levels, **: statistically significant difference at 0.01 levels according to the Ducan test Table 4.15 shows that the density of microalgae appeared at the beginning of the tillering stage (the first stage) that was 3.0 to 5.9 times higher than that in the panicle and ripening stages (p < 0.05) In particular, the Bacillariophyta contributed the highest density and mainly at the beginning of the tillering stage but the Cyanobacteria contributed the least density Table 4.15: Density of planktonic microalgae (Individual/L) appeared during the growth stages of rice plants in three rice crops Stage 1st stage n Bacillariophyta Chlorophyta Euglenophyta Cyanobacteria 27 28,117a 6,009a 2,365b 2nd stage 27 4,691b 1,131b 10,752a 3,714a Total 33.527a 4,682a 23,035ab 3rd stage 27 833b 310b 2,089b 381b 11,035b 4th stage 27 2,207b 288b 5,308ab 503b 5,728b 5.17** 9.41** 2.96* 7.00** 3.18* F value Note:*: significant difference at 0.05 levels, Ducan test ** : statistically significant difference at 0.01 levels according to the 28 4.2.2.2 Species diversity and density of benthic microalgae in the Winter-Spring rice of 2016-2017 and the Summer-Autumn rice of 2017 a Species diversity of benthic microalgae in two rice crops Table 4.16 shows that there were 157 taxa of benthic microalgae belonging to 63 genera, 31 families, 14 orders and three phyla (Chlorophyta, Bacillariophyta, and Cyanobacteria), in which Chlorophyta had a number of species the highest, followed by Bacillariophyta and Cyanobacteria However, there was no great alteration in the number of species and the structure of species composition between the two crops There were 114 taxa in the Winter-Spring rice and 110 taxa in the Summer-Autumn rice The number of species presented in the two crops was a great difference compared with the total number of species due to environmental conditions between two rices Studies on the appearance of benthic microalgae in fields according to rice crops have not yet been published Thus, there is no scientific basis to explain why this species only appears in the Winter-Spring rice but not in the Summer-Autumn rice and vice versa This research has identified a species benthic of Cyanobacteria Calothrix aeruginosa, which lives in the bottom of topsoil in the Winter-Spring rice, is capable of fixing nitrogen in the soil Table 4.16: The structure of species composition of benthic microalgae in the rice fields in Winter-Spring and Summer-Autumn rices Phylum Order Family Genus Taxa Percentage of taxa (%) Bacillariophyta 15 51 32.5 Chlorophyta 18 43 98 62.4 Cyanobacteria 5 5.1 14 31 63 157 100 Total b Density of benthic microalgae in two rice crops Table 4.17 shows that the total density of benthic microalgae appeared in the growth stages of rice plants in the Winter-Spring rice was a significant difference (p < 0.05) Total algal density of the second stage (at the end of tillering) was the highest, followed by the first stage (the beginning of tillering), the third stage (panicle initiation) and the fourth stage (ripening) The Bacillariophyta appeared with high density at the end of the tillering stage Microalgal density at the beginning of the tillering stage was lower than that at the end of tillering stage because the farmers sprayed herbicide such as Michelle 62EC and Cantanil 550EC after six days sowing The butachlor active of Michelle 62EC has the ability to kill green algae such as Pseudokirchneriella subcapitata and Desmodesmus subspicatus and the propanil active of Cantanil 550EC inhibits the growth of genera of Cyanobacteria such as Anabaena and Nostoc, or inhibit nitrogenase of Cyanobacteria in floodplains However, the density of benthic microalgae at the beginning of the tillering stage was higher than that in the panicle stage because the farmers sprayed insecticide such as Confitin 75EC, Indosuper 150SC and Tungcydan 30EC before fertilizing one day The activities of them are highly toxic to aquatic organisms and algae On the other hand, in the third stage, which is panicle stage (preparing to flower), the rice concentrates on nourishment, especially rice concentrates to absorb nutrients such as nitrogen and phosphorus, resulting in nitrogen and phosphorus of water in the field decreases quickly, so that algae absorb the nutrients less 29 The total density of benthic microalgae in the ripping stage was lower than that in the panicle stage, although the clinging time of algae in the fields at the ripping stage (44 days) was longer than the panicle stage (10 days) about 33 days The main reason is that blast disease attacked the rice plants after the third fertilizing so farmers sprayed Fuan 40EC and Trizole 75WP so that they would kill them Besides, Tilt Super 300EC was sprayed to prevent blemish disease Table 4.17: Density of benthic microalgae (103 individuals/m2) in the rice field in the Winter-Spring and the Summer-Autumn rices Rice crop WinterSpring Stage Chlorophyta Cyanobacteria Total density 1st stage 9,929±6,199b 2,175±393 117±14b 12,221±6,098b 2nd stage 20,500±3,733a 2,083±260 167±72a 22,750±3,929a 3rd stage 2,933±252c 1,328±79 72±9,62c 4,333±333c 294±174d 3,028±1,075 65±40d 3,387±1,024d 4th stage 9.97* 9.97* 6.65ns 9.56* 1st stage 1,264±591c 2,589±833 86±33 3,939±1,413b 2nd stage 14,959±2,068a 1,220±202 54±14 16,233±1,900a 3rd stage 437±8.37d 734±246 57±18 1,228±248d 4th stage 1,467 ±1,161b 1,383±951 37±3 2,887±2,114c 8.23* 7.46ns 6.13ns 8.23* Asymp Sig SummerAutumn Bacillariophyta Asymp Sig Note: In the same column, the same followed digits are not significantly different at the significance level of 0.05, ns: no significant difference at 0.05 levels, *: statistically significant difference at 0.05 levels according to the Kruskal – Wallis test Similar to the Winter-Spring rice, Table 4.17 shows that the algal density appeared in the growth stages of rice plants in the Summer-Autumn rice was significantly different (p < 0.05), in which the density of diatoms of the second stage accounted for 92.2%, but the total density of green and blue-green microalgae accounted for only 7.8% of the total density The algal density of the first stage was lower than the second stage because the farmers sprayed herbicide such as Dietmam 360EC and Cantanil 550EC after five days sowing The algal density of the third stage was the lowest because of spraying insecticide that has the ability to kill microalgae These are Chess 50WG, Indosuper 150SC, Tungperin 10EC and Acfubim 800WP The microalgal density of the fourth stage was higher than the third stage (p < 0.05) because after sixty days sowing (after collecting the samples of the third stages), the rice plants were attacked by leaf pests, hoppers, blast disease, and leaf blight, so the farmers sprayed insecticide such as Tungperin 10EC, Chess 50WG, Amistar Top 325SC and New Kasuran 16.6WP However, the time between spraying and collecting microalgae samples was 16 days Farmer’s irrigation water for rice helped to dilute the insecticide and drain the field ditches instead of the numbers of microalgae were supplemented to the field through irrigation water Besides, phosphorus fertilizer only applied in the fourth stage (1.07 kgP2O5/1,000 m2 areas) but not applied in the third stage, but phosphorus is more important than nitrogen Table 4.18 shows that the density of benthic microalgae in the third stage in the Winter-Spring rice was significantly higher than the Summer-Autumn rice (p < 0.01) 30 Because the weather in the Summer-Autumn rice is rainy, average rainfall was 267 mm/crop (NASA, 2019), high humidity but little sunshine that has created very favorable conditions for disease and pest attack, while the weather in the Winter-Spring rice is very good, it is good sunshine, but little rain and humidity not too high Thus, rice plants should be less sick and pest attack On the other hand, the amount of nitrogen fertilizer and phosphorus applied to the rice in the third stage of the Winter-Spring rice was higher than that of the Summer-Autumn rice (Table 3.3), so the amount of nitrogen and phosphorus provided with microalgae in the Winter-Spring rice was higher Table 4.18: Comparison of algal density (103 individuals/m2) between the Winter-Spring and the Summer-Autumn rices in the same growth stage of rice plants Stage Winter-Spring rice Summer-Autumn rice t value 1st stage 12,221±6,098 3,939 ±1,413 2.29ns 2nd stage 22,750±3,929 16,233±1,900 2.59ns 3rd stage 4,333±333a 1,228±248b 13.0** 4th stage 3,387±1,024 2,887±2,114 0.20ns Note: In the same row of the same digits, the difference is not statistically significant at the significance level of 0.05; **: difference at significance level 0.01; ns: not significant difference at 0.05 levels according to two independent tests (T-test) The study results also showed that the total average density of microalgae appeared in each developmental stage of the rice in the Winter-Spring rice (10,673±2,846×103 individuals/m2) was higher than the Summer-Autumn rice (6,071±1,419×103 individuals/m2), in which the density of diatoms contributes the highest, followed by green microalgae and blue-green microalgae 4.2.2.3 Presence of microalgae phyla help to improve the environmental soil First, the role of nitrogen fixation of Cyanobacteria; the research has determined six species of Cyanobacteria that are capable of fixing nitrogen in rice fields, including five species of planktonic Cyanobacteria and one species of benthic Cyanobacteria However, Anabaena oscillarioides appeared with high density at the beginning of the tillering stage, which fixed nitrogen from the atmosphere to the soil The content of nitrogen fixed by Cyanobacteria in natural soil was 14 kgN/ha (De and Mandal, 1956 cited by FAO of the United Nations, 1981) During the appropriate season, The content of fixed nitrogen was from 20 to 30 kgN/ha (Kaushik, 1994; Dang Dinh Kim and Dang Hoang Phuoc Hien, 1999) As applying phosphorus fertilizer to the soil, the content of fixed nitrogen increased from 18 to 69 kgN/ha The content of fixed nitrogen of the soil increased from to 8% after 30 days of culturing fix-nitrogen species (Nguyen Xuan Thanh et al., 2009) or increased 31.6% compared with control treatments (Yagya and Shreeti, 2012) In addition, the spraying of Cyanobacteria strains on rice plants also improved chemical properties of the soil such soil pH, humus, total nitrogen and available nitrogen corresponding to 10.7%, 4.83%, 5.56% and 8.92% compared with the control treatment (Nguyen Dinh San, 2015) and the content of organic carbon of the soil increased 68.7% compared with the control treatment (Singh, 1961 cited by Roger and Kulasooriya, 1980) If the farmers use them effectively, the weight of chemical fertilizers applied to rice can reduce from 15 to 30% of total weight (Kaushik, 1994, Dang Dinh Kim and Dang Hoang Phuoc Hien, 1999) 31 Next, depending on the specific algae phyta that the kinds of nutrients returned differently for the soil, for example, Cyanobacteria supply more nitrogen for the soil than the green microalgae and diatoms Meanwhile, green algae provide higher lipid content than diatoms (Dao Thanh Son et al., 2014) but diatoms provide high carbohydrate content When microalgae die, the nutrients of them can be returned for the soil, so the nutrients improve the soil environment 4.2.3 Biomass of planktonic and benthic microalgae to improve the soil environment 4.2.3.1 Biomass of planktonic and benthic microalgae in the Autumn-Winter rice of 2016 Table 4.19 shows that the biomass of planktonic microalgae in the Autumn-Winter rice ranged from 0.10 to 3.68 (mg/L) and there were significant differences between fertilizing times, with p < 0.05 The biomass of planktonic microalgae of the first stage was the highest because the rice leaf canopy on the field surface was still sparse, and the weather was sunny, so the amount of light transmitted on the field surface helped algae photosynthesis increase biomass This is appropriate because the result of observing in the field showed that a lot of pieces of yellow brown algae appeared on the field surface (Figure 4.11) Figure 4.11: Yellow brown microalgae pieces appeared on the field surface in Autumn-Winter rice of 2016 The biomass of planktonic microalgae in the first stage was statistically similar to the second stage but the biomass of benthic microalgae in the first stage was lower than the second stage because the farmers sprayed Tungsai 700WP, Michelle 62EC and Cantanil 550EC insecticide to kill yellow snail one day before sowing, and six days before sowing The niclosamide of Tungsai 700WP insecticide is effective at killing green microalgae (Chlorella vulgaris) and a lethal dose of 50% of green microalgae is ppm for 48 hours (Karuppasamy et al., 2018) The butachlor and propanil of Michelle 62EC and Cantanil 550EC insecticides also killed the green microalgae Pseudokirchneriella subcapitata and Desmodesmus subspicatus (Park et al., 2009) and inhibited the growth of Anabaena gena (Ibrahim, 1972 and Wright et al., 1977 were cited by Pingali and Roger, 1995) and the activity of nitrogen-fixing nitrogenase in Nostoc calcicola (Pandey, 1985) The biomass of planktonic and benthic microalgae in the third stage and the fourth stage gradually decreased because of the effects of rain The average rainfall in October in 2016 was 320 mm/month (NASA, 2019), water level in the field in the third and the fourth stages were 4.4 and 6.14 cm, respectively (Table 4.11) In addition, the height and number of branches of rice plants have reached the maximum quantity and there is a rising stalk (Nguyen Van Bo et al., 2016), with low light intensity, so the amount of light transmitted to the field surface is less than the limited photosynthesis speed of algae The biomass of benthic microalgae was lowest in the fourth stage, because the rice leaf canopy on the field was dense, the amount of light shining on the field was very 32 little, and the rain lasted from the third to the fourth fertilization The main reason is that farmers sprayed insecticides on the rice plants to kill leaf pests, hoppers, blast disease, and leaf blight before fertilizing at the fourth stage These have killed many species of algae, so the algal biomass decreased Table 4.19: Biomass of planktonic and benthic microalgae in rice fields in three rice crops Autumn-Winter Rice Stage Number of sample Planktonic microalgae (BR, mg/L) Range Average Range Average 1st stage 0.228-3.68 1.61±1.15a 290-746 540±231bc 2nd stage 0.125-3.68 1.02±1.14ab 792-1,423 1,172±335a 3rd stage 0.100-1.45 0.474±0.400b 785-1,069 912±144ab 4th stage 0.264-1.60 0.775±0.477ab 114-316 234±106c 2.78* Winter-Spring F value 10.3** 1st stage 0.096-14.0 3.92±4.77a 1,345-2,210 1,738±438b 2nd stage 0.186-1.52 1.97±2.17ab 2,169-2,826 2,495±328a 3rd stage 0.147-1.29 0.706±0.034b 768-1,337 1,048±159c 4th stage 0.065-0.86 0.367±0.216c 244-556 416±159d 3.38* F value Summer-Autumn Benthic microalgae (BĐ, mg/m2) 23.7** 1st stage 1.43-43.6 13.0±16.6a 669-1,366 1,124±395b 2nd stage 1.10-2.21 1.87±0.381b 1,809-2,167 2,038±199a 3rd stage 1.13-1.79 1.40±0.190b 180-328 233±82d 4th stage 1.36-2.00 1.62±0.241b 187-632 360±238c 4.22* F value 9.46* Note: In the same row of the same digits, the difference is not statistically significant at the significance level of 0.05; **: difference at significance level 0.01; ns: not significant difference at 0.05 levels according to T-test 4.2.3.2 Biomass of planktonic and benthic microalgae in the Winter-Spring rice of 2016-2017 The biomass of planktonic microalgae in the first stage of the Winter-Spring rice was significantly different from the third and the fourth stages, with p < 0.05 (Table 4.19) The main reason is the rice leaf canopy on the field surface was sparse and the weather was sunny This result is suitable for the algal quantitative results; the total algal density in the first stage was 69,295 individuals/L The density of diatom phyta accounted for 89.2% and the others accounted for 10.8% of total density (Table 4.15) However, Table 4.19 shows that benthic microalgae biomass in the second stage was significantly different from the others (p < 0.01) The biomass of benthic microalgae of the second stage was higher than the first stage because the farmers sprayed Michelle 62EC and Cantanil 550EC herbicides in six days after sowing As explained above, the butachlor and propanil was also capable of killing green microalgae and inhibiting the growth of Cyanobacteria This result is suitable for the algal quantitative results, the total 33 density of benthic microalgae of the second stage (22,750 × 103 individuals/m2) was significantly higher than the first stage (12,221 × 103 individuals/m2) (Table 4.17) Table 4.12 shows that the biomass of planktonic microalgae in the second stage was equivalent to that of the third stage (p > 0.05), and the density of planktonic microalgae of two stages was statistically similar (p > 0.05) However, the average density of planktonic microalgae of the second stage accounted for 84.8% (22,746 individuals per 26,820 total individuals), the density of Chlorophyta and Cyanobacteria were relatively low 4.28% (1,148 individuals per liter) Because genena of Euglenophyta, especially Euglena, mostly have heterotrophic life, about 1,000 taxa belonging to this genena often live in rice fields, have fertilized chemical fertilizers (Columbia Electronic Encyclopedia, 2019) In addition, this study applied indirect fluorescence methods to determine chlorophyll concentration (TCVN 6662: 2000), even though the density of Euglenophyta is high, the measured biomass may not be high Similar to the Autumn-Winter rice, the biomass of planktonic microalgae of the fourth stage tended to decrease strongly because the rice leaf canopy on the field surface was dense and the amount of chemical fertilizers applied to the rice plants was the lowest The pH value of water in the field (Figure 4.5) suddenly decreased, algae did not adapt in time so it was inhibited and unable to reproduce Therefore, the process of creating oxygen from algae photosynthesis was also low, the DO concentration of this stage (3.39 mg/L, Figure 4.9) was the lowest Similar to the Autumn-Winter rice, the biomass of benthic microalgae of the third stage was the lowest (p < 0.05) because the rice leaf canopy on the field surface was dense The nutrients of water in the field decreased significantly due to the rapid absorption of the rice plant The farmers sprayed insecticides (such as Confitin 75EC, Indosuper 150SC and Tungcydan 30EC) to the rice plants before fertilizing at this stage The biomass of benthic microalgae of the fourth stage was lower than the third stage (Table 4.19) Because the farmers only applied nitrogen fertilizer with low amounts but did not apply phosphorus fertilizer in the fourth stage, the rice leaf canopy on the field surface was dense so sunlight transmits on the field surface little 4.2.3.3 Biomass of planktonic and benthic microalgae in the Summer-Autumn rice of 2017 Similar to the previous two crops, the biomass of planktonic microalgae of the first stage was significantly different from the others (Table 4.19) Because the density of Chlorophyta and Cyanobacteria of this stage was the highest, but they mostly have autotrophic life, photosynthetic pigments are mainly chlorophyll-a, and chlorophyll-b (Duong Duc Tien, 1996; Vu Ngoc Ut and Duong Thi Hoang Oanh, 2013) The biomass of planktonic microalgae of the first stage was higher than the second stage (p < 0.05) because the density of green algae of the first stage (5,618 individuals/L) was significantly different from the second stage (1,201 individuals/L), with p < 0.01 However, the benthic microalgae biomass of the second stage was significantly different from the others (p < 0.05) The reason is explained similar to the previous two crops because of the influence of weather, the rice leaf canopy and the weight of chemical fertilizers for rice plants The biomass of planktonic microalgae of the third stage was the lowest due to hoppers and leaf pests attacking the rice plants Thus, the farmers sprayed some kinds of insecticides such as Chess 50WG, Indosuper 150SC, Tungperin 10EC and Acfubim 34 800WP in one day before fertilizing The pymetrozine active of Chess 50WG is capable of killing 50% of the number of green algae Pseudokirchneriella subcapitata with the concentration is above 100 mg/L for 72 hours (Syngenta Switzerland, 2013b) The permethrin active of Tungperin 10EC affects the growth, photosynthesis and de-acetylene ability of two green algae species (Chlorella pyrenoidosa and Scenedesmus quadricauda) and three species of Cyanobacteria (Anabaena spp.), in which Cyanobacteria was more sensitive than Chlorophyta (Stratton and Corke, 1982) In addition, the biomass of benthic microalgae of the third stage was also the lowest because hoppers and leaf pests attracted the rice plants Besides, the farmer did not apply phosphorus fertilizer at this stage, but only apply nitrogen and potassium fertilizers (Table 3.3) To increase biomass, algae need to be provided with phosphorus and nitrogen fertilizers but phosphorus is more necessary than nitrogen and it is a limit factor the growth of algae (Round, 1975) 4.2.3.4 Biomass of planktonic microalgae in the canal Xa Mach canal is a source of water supplement for the fields inside of the full-dyke systems Table 4.20 shows that the biomass of planktonic microalgae in the canal in the Autumn-Winter, the Winter-Spring and the Summer-Autumn rices averaged 0.198, 0.272 and 0.583 mg of dry biomass per liter per rice crop, respectively They were equal to 2.93, 2.95 and 9.77 kg of dry biomass per hectare per rice crop; with the biomass of planktonic microalgae in the canal was 15.7 kg of dry biomass per hectare per year Besides, the biomass of planktonic microalgae in the rice fields was 55 kg of dry biomass per hectare per year Thus, total biomass of planktonic microalgae in both rice fields and canal was 70.7 kg of dry biomass per hectare per year equal to 0.071 tons of fresh biomass per hectare per year with the amount of water in algal biomass accounts for 82% of total biomass Table 4.20: The biomass of planktonic microalgae in both rice field and canal (BPD, kg/ha) provided with the soil every year (dry biomass) Stage n Autumn-Winter rice Winter-Spring rice Summer-Autumn rice BTN BR BTN BR BTN BR 0.288 2.35 0.687 7.08 4.63 23.8 0.787 1.28 0.644 3.64 2.97 5.61 0.236 1.26 1.10 1.33 1.24 2.59 1.62 2.74 0.515 0.418 0.922 2.96 Total stage 12 2.93 7.62 2.95 12.5 9.77 34.9 Biomass/crop (BPD) 12 10.6±1.24 year 36 st nd rd th stage stage stage stage 15.4±2.95 44.7±11.7 70.7±15.9 Note: BTN: biomass of planktonic microalgae in canal; BR: biomass of planktonic microalgae in rice field, BPD = BTN + BR 4.2.3.5 Total biomass of planktonic and benthic microalgae provide for annual rice field Table 4.21 shows that the biomass of benthic microalgae was 123 kg of dry biomass per hectare per year, of which that in the Winter-Spring rice accounted for 46.3%, followed by the Summer-Autumn rice (30.5%) and the Autumn-Winter rice (23.2% of total benthic microalgae biomass) 35 Table 4.20 and Table 4.21 show that total biomass of microalgae provided with the rice fields annually was 194 kg of dry biomass per hectare per year equal to 1.08 tons of fresh biomass per hectare per year Benthic microalgae biomass was 1.74 times the planktonic microalgae biomass This is suitable with the study of Cahoon, Safi in 2010, the biomass of benthic microalgae is higher than planktonic microalgae, and it may be four times the planktonic microalgae Table 4.21: The biomass of benthic microalgae in rice field (BĐ, kg/ha) provided with the soil every year (dry biomass) Stage n Autumn-Winter rice Winter-Spring rice Summer-Autumn rice 1st stage 5.40±2.31 bc 17.4±4.38 b 11.2±3.95 b 2nd stage 11.7±3.34 a 24.9±3.28 a 20.4±2.00 a 3rd stage 9.12±1.45 ab 10.5±2.84 c 2.33±0.825 d 4th stage 2.34±1.06 c 4.16±1.59 d 3.61±2.39 c 10.3** 23.7** 9.46* 28.6±8.16 57.0±12.1 37.6±9.14 F value one rice one year 12 123±29.4 Note: In the same column of the same digits, the difference is not statistically significant at the significance level of 0.05, **: difference at significance level 0.01 according to Duncan tests 4.2.4 Factors affect planktonic microalgae biomass 4.2.4.1 Effect of the rice leaf canopy on planktonic microalgae biomass Table 4.22 shows that planktonic microalgae biomass of the treatment without rice but with fertilizer (NL2) was significantly different from that of the treatment without rice and with fertilizer (NL1) (p < 0.05) The algal biomass of NL2 treatment was 5.95 times that of NL1 treatment and the algal biomass increased 161 mg/L, equal to 83.2% of the total biomass because of applying nitrogen and phosphorus fertilizers to rice plants, algae absorbed nutrients of chemical fertilizers to increase biomass Table 4.22: The biomass of planktonic microalgae (mg/L) of treatments without rice (NL1 and NL2) in the pot experiment in the Winter-Spring rice of 2017-2018 Experimental treatment n Range Average NL1 (Without rice plant, 0% Fertilizer) 20.8-56.5 32.6±16.3b NL2 (Without rice plant, with 100% Fertilizer) 89.2-398 194±134a |Z| value 0.029* CV (%) 7.89 Note: In the same column of the same digits, the difference is not statistically significant at the significance level of 0.05, *: difference at significance level 0.05 according to Mann-Whitney tests 4.2.4.2 Effect of the weight of chemical fertilizer on planktonic microalgae biomass Table 4.23 shows that the total algal biomass between four treatments with rice and with fertilizer (such as NL3, NL4, NL5 and NL6 treatments) was a significant difference (p < 0.01) The algal biomass of NL5 treatment (with 100% fertilizer) was the highest, followed by NL6 (with 130% fertilizer), NL4 (with 70% fertilizer) and NL3 treatment (0% fertilizer) Fertilizer contributed to increasing microalgal biomass from 1.79 to 3.66 times However, the amount of fertilizers and algal biomass was not proportional to each 36 other; it means that the algal biomass of NL6 treatment (with 130% fertilizer) was lower than that of NL5 treatment (with 100% fertilizer) The algal biomass of NL5 and NL6 treatment is 106 mg/L and 74.6 mg/L, respectively Because the concentration of phosphate in the water exceeds 18 mg/L, it can affect the growth of microalgae or cause inhibition of them (Vu Ngoc Ut and Duong Thi Hoang Oanh, 2013) Comparison of algal biomass of the treatment without rice and with fertilizer (NL1) and the treatment with rice but without fertilizer (NL3), the results showed that the algal biomass of NL1 (Table 4.22) was 1.13 times that of NL3 (Table 4.23) It means that the rice leaf canopy limited sunlight transmitting the water surface, so it can reduce the photosynthesis activity of microalgae equal to a reduction of 11.4% of the total biomass In general, the rice leaf canopy and the chemical fertilizers all affected the algal biomass Table 4.23: The biomass of planktonic microalgae (mg/L) of treatments with rice in the pot experiment in the Winter-Spring rice of 2017-2018 Experimental treatment with rice n Range Average Ratio of algal biomass of the treatment with fertilizer/no fertilizer NL3 (0% Fertilizer) 26.3-31.5 28.9±2.50d 1.00 NL4 (70% Fertilizer) 47.7-58.1 51.6±4.50c 1.79 NL5 (100% Fertilizer) 102-110 106±6.14a 3.66 NL6 (130% Fertilizer) 66.5-87.6 74.6±9.92b 2.58 F value 9.85** CV (%) 5.10 Note: In the same column of the same digits, the difference is not statistically significant at the significance level of 0.05, **: difference at significance level 0.01 according to Duncan tests 4.3 Evaluating the contribution rate of total nitrogen, phosphorus and potassium of deposited sediment and microalgae 4.3.1 Nutrients of microalgae provided with the soil Among the three NPK components of microalgae, total potassium was the highest content, followed by total phosphorus and nitrogen, with the amount of NPK provided with the soil equal to 1.26 kgK/ha; 0.465 kgN/ha; and 0.197 kgP/ha every year (Table 4.24 and Table 4.25) Table 4.24: The content of total NPK of planktonic and benthic microalgae (dry biomass) Nutrients of microalgae Total N (mgN/kg) Total P (mgP/kg) Total K (mgK/kg) Planktonic microalgae in rice field 1,647 1,146 6,028 Benthic microalgae in rice field 2,958 954 6,554 Planktonic microalgae in canal 693 1,062 7,665 Average content of microalgae 2,303 1,050 6,291 The amount of total phosphorus and potassium of microalgae provided with the rice soil in the Summer-Autumn rice was the highest, followed by the Winter-Spring and the Autumn-Winter rices; in contrast, the amount of total nitrogen of the Winter-Spring rice was higher than two rices The amount of total NPK of benthic microalgae was higher than that of planktonic microalgae, equal to 4.02, 1.86 and 2.43 times, respectively 37 Table 4.25: The amount of total NPK of planktonic and benthic microalgae provided with the rice soil (dry biomass) Total N (kgN/ha) Rice crop Total P (kgP/ha) Total K (kgK/ha) PD D TN Total PD D TN Total PD D TN Total TD 0.013 0.084 0.002 0.099 0.009 0.027 0.003 0.039 0.046 0.187 0.022 0.256 DX 0.021 0.168 0.002 0.191 0.014 0.054 0.003 0.072 0.075 0.373 0.023 0.471 HT 0.058 0.111 0.007 0.175 0.040 0.036 0.010 0.086 0.210 0.246 0.075 0.531 Total 0.092 0.363 0.011 0.465 0.063 0.117 0.016 0.197 0.331 0.806 0.120 1.26 Note: PD: planktonic microalgae in rice field, D: benthic microalgae in rice field, TN: planktonic microalgae in canal, TD: Autumn-Winter rice, DX: Winter-Spring rice, HT: Summer-Autumn rice 4.3.2 The contribution rate of total nitrogen, phosphorus and potassium of deposited sediment and microalgae for the soil Comparison of the total NPK content of deposited sediment and rice straw (Table 4.26) showed that the total phosphorus content of sediment was 4.82 times that in rice straw, but the total nitrogen and potassium content of rice straw was higher than that of sediment equal to 1.63 and 1.33 times The content of total phosphorus of microalgae was 1.74 times that of rice straw In summary, deposited sediment was dominant about total phosphorus and rice straw was dominant about total nitrogen and potassium Table 4.26: The content of total NPK of sediment, microalgae and rice straw (dry weight/dry biomass) Component Total N (gN/kg) Total P (gN/kg) Total K (gK/kg) Sediment(1) 3.26 2.41 14.2 Microalgae 2.30 1.05 6.29 Rice straw(2) 5.30 0.500 18.9 (1) Notes: : analyzed the result of total NPK content in sediment inside of the full-dyke (Content 1.1); to Abou-El-Enin et al (1999) was cited by Tran Sy Nam (2016) (2) : according The biomass of rice straw embedded in the soil was calculated based on the biomass of rice straw (ton/year) and the percentage of the rice area was buried rice straw in the soil (18.8 × 10.9% = 2.049 kg/ha/year) The biomass of rice straw (Brice-straw, ton/year) = Rice yield × Ratio of rice straw and rice yield × number of crops/year = 5.5 × 1.14 × = 18.8 (tons/ha/year) According to Tran Sy Nam et al (2014), the ratio of soil area was buried rice straw accounts for 6.7% and 26.1% equal to in the Summer-Autumn rice and in the Winter-Autumn rice However, the rice straw in the Winter-Spring rice was mainly burned Thus, the percentage of rice area buried by rice straw every year accounted for 10.9% of total biomass Table 4.27 shows that the rice soil inside of the full-dyke could receive the amount of total NPK of microalgae was 0.465 kgN/ha/year, 0.197 kgP/ha/year and 1.26 kgK/ha/year, respectively The amount of total NPK of microalgae was much lower than that of rice straw, but microalgae was regularly a supplementary source of nutrients for the soil In general, it was found that nutrients of microalgae play an extremely important role and help quickly to return to the soil because nutrients of microalgae are usually in digestible form, so rice plants easily absorb in a short time On the other hand, the burial of fresh rice straw in wetlands easily causes organic poisoning of rice roots due to toxic 38 substances such as total organic acids and hydrogen sulfur gas were generated from the anaerobic decomposition of rice straw (Nguyen Thanh Hoi et al., 2009) The study has not yet estimated the amount of total nitrogen of microalgae really provided with the soil because it has not yet calculated the amount of total nitrogen was fixed by Cyanobacteria Therefore, the amount of total nitrogen of algal biomass was about 30 times lower than the amount of real nitrogen This is the basis to help explain why it has been nearly 20 years since the full-dyke in An Giang province The rice plants were grown continuously in three crops per year but have not shown signs of chemical soil of degradation, instead of total NPK content of the soil inside of the full-dyke were significantly different from that outside of the full-dyke (Nguyen Huu Chiem et al., 2017) Table 4.27: Weight/Biomass and total NPK content (kg/ha) of sediment, microalgae and rice straw provided with the soil (dry weight/dry biomass) Component Total weight/biomass (kg/ha) Total N (kgN/ha) Total P (kgP/ha) Total K (kgK/ha) Content % Content % Content % 4,430 14.4 3.85 10.7 3.95 62.9 40.3 194 0.465 0.124 0.197 0.073 1.26 0.807 Rice straw 2,049 10.9 2.90 1.02 0.38 38.7 24.8 Total 6,673 25.8 6.99 12.1 4.48 104 66.7 Sediment Microalgae Note: Percentage (%) compared with weight of chemical fertilizer There is a symbiotic relationship between microalgae and rice plants because microalgae can convert ammonia into nitrate for its biomass, while rice plants prefer to absorb nitrate for growth, which means microalgae provide nitrate for rice, so it needs less fertilizer (Mazur, 2016) If the farmers utilize three sources of nutrients supplemented by deposited sediment, microalgae and rice straw, the soil would receive the nutrients of them with the amount of total nitrogen, phosphorus and potassium was equal to 66%, 6.87% and 4.41% of total chemical fertilizers (N, P2O5 and K2O), respectively Therefore, it is possible to save the cost of buying fertilizers, and can take advantage of other resources such as organic matter, Si, Ca, Mg, Mn, etc of deposited sediment and microalgae, to improve the rice soil inside of the full dyke systems Therefore, both deposited sediment and microalgae are a valuable source of nutritional supplement for the soil CHAPTER 5: CONCLUSIONS AND SUGGESTIONS 5.1 Conclusions The weight of deposited sediment outside of the full-dyke in An Giang province was five times than inside of the full-dyke systems The weight of deposited sediment accumulated in the soil for three years or more, the nutrients of the sediment have contributed to improve the content of organic carbon and total phosphorus of the soil after harvest, and increased 1.5 and 1.29 times compared with the soil before planting, respectively There were the presences of 445 species of planktonic and benthic microalgae in the rice fields, belong to four phyla (Bacillariophyta, Chlorophyta, Euglenophyta and 39 Cyanobacteria), and in which Chlorophyta is the most diverse in species composition structure Anabaena gena belongs to Cyanobacteria and has planktonic life that appeared in high density at the beginning of the tillering stage of the Summer-Autumn rice of 2017 It is a source of supplementation of the bio-nitrogen for the soil Annually, microalgal biomass estimated 1.08 tons of fresh biomass per per year, the content of total phosphorus of microalgae was 2.1 times than that of rice straw, and when they died, it can be returned the nutrients for the soil The contribution rate of total NPK of deposited sediment was higher than that of microalgae The rice soil would receive an amount of nutrients equal to 3.98%, 4.03% and 41.1% of the chemical fertilizers (N, P2O5 and K2O), respectively It shows that the deposited sediment and microalgae are an available nutrient for the rice soil Therefore, the deposited sediment and microalgae are important for rice production in the flood area of An Giang province in particular and in the Mekong Delta in general 5.2 Suggestions In the Summer-Autumn rice, during the tillering stage, Cyanobacteria with heterocysts appeared with relatively high density, so it is necessary to have appropriate solutions to promote their ability to fix nitrogen into the rice soil It is necessary to continuously study species composition of benthic microalgae in the Autumn-Winter rice to modify the data set of species composition in the Mekong Delta It is necessary to study continuously on the total nutrients provided from algal biomass, amount of nitrogen fixed by Cyanobacteria; the total carbon organic and macronutrients accumulated in the soil It is recommended to discharge floods in the Autumn-Winter rice so that the rice field could receive deposited sediment and wash away the toxins and utilize effectively the nutrients of deposited sediment and microalgae 40 ... Cong Khanh, Pham Van Toan and Nguyen Huu Chiem, 2017 Study on the quantity and nutrients content of sediment in the full-dyke and semidyke systems in An Giang province Journal of Science, Can Tho... (Kaushik, 1994, Dang Dinh Kim and Dang Hoang Phuoc Hien, 1999) CHAPTER 3: METHODOLOGY 3.1 Research place and time The research was carried out in four districts of An Giang province and An Giang University... Cyanobacteria genera can help to increase the rice yields There are Tolypothrix (in Japan), Anabaena (in India, Nepal, Iran, and Vietnam), Calothrix (in Vietnam), Nostoc (in Bangladesh and Vietnam),