Digital image analysis of duckweed growth in swine wastewater after anaerobic treatment

VIETNAM NATIONALa UNIVERSITY, HANOI VIETNAM JAPAN UNIVERSITY DINH THI TO UYEN DIGITAL IMAGE ANALYSIS OF DUCKWEED GROWTH IN SWINE WASTEWATER AFTER ANAEROBIC TREATMENT MASTER'S THESIS Hanoi, 2018 VIETNAM NATIONAL UNIVERSITY, HANOI VIETNAM JAPAN UNIVERSITY DINH THI TO UYEN DIGITAL IMAGE ANALYSIS OF DUCKWEED GROWTH IN SWINE WASTEWATER AFTER ANAEROBIC TREATMENT MAJOR: ENVIRONMENTAL ENGINEERING RESEARCH SUPERVISOR: Assoc Prof Cao The Ha Prof Jun Nakajima Prof Satoshi Soda Dr Nguyen Thi An Hang Hanoi, 2018 ACKNOWLEDGMENTS First and foremost, I would like to express my gratitude to my supervisor, Associate Professor Cao The Ha and Professor Jun Nakajima who have been more of a friend to me than a supervisor I find myself lucky to have the opportunity to study and work with them They have been always there to guide me and help me out with great patience not only for academic but also for daily life I gratefully acknowledge the financial support from Vietnam Japan University and from Associate Professor Cao The Ha With their support, I can put all my focus on the researcher Many thanks to Professor Satoshi Soda for his keen guidance in preparing this thesis during my internship in Japan In addition, special thanks to Dr Nguyen Thi An Hang for the valuable working experiences She is instilled on me a active passion for research from the beginning to the end Her admirable spirit of scientific rigor is also impressive It is a pleasure for me to show my gratitude to Dr Vu Ngoc Duy and all members of Environmental Technology Laboratory in Center for Environmental Technology and Sustainable Development, who instructed, supported, encouraged me during the completion of the thesis I would like to thank teachers in Environmental Engineering Program from Vietnam Japan University who gave me essential knowledge of environmental major Last but not least, I would like to thank my family, my classmates and my friends for their unfailing love and support throughout this process to complete my master’s degree Hanoi, June 15th, 2018 Dinh Thi To Uyen i TABLE OF CONTENTS ACKNOWLEDGMENTS i TABLE OF CONTENTS ii LIST OF FIGURES v LIST OF TABLES vi LIST OF ABBREVIATIONS vii INTRODUCTION CHAPTER LITERATURE REVIEW 1.1 Swine wastewater and environmental impacts in Vietnam 1.1.1 The status of livestock development in Vietnam 1.1.2 The status of swine wastewater after anaerobic treatment in Vietnam 1.1.3 Environmental impacts of animal waste 1.2 Swine wastewater treatment technologies in the world and Vietnam 1.2.1 Popular treatment technologies 1.2.2 Phytoremediation 1.3 Duckweed as a potential plant for phytoremediation of swine wastewater treatment after biogas 10 1.3.1 General characteristics of duckweed 10 1.3.2 Factors affecting the growth of duckweed 12 1.3.3 The ability of duckweed in treating pollution 13 1.4 Growth evaluation using Digital Image Analysis processing 14 1.4.1 Popular methods to evaluate plant growth 14 1.4.2 Assessing duckweed growth by measuring frond area with software 15 CHAPTER MATERIALS AND METHODS 17 2.1 Materials and instruments 17 ii 2.1.1 Chemicals and instruments 17 2.1.2 Duckweed 17 2.1.3 Wastewater 18 2.2 Methods 19 2.2.1 Field survey and sampling 19 2.2.2 Experimental setup 20 2.2.2.1 Study on the growth of Lemna Minor in Hyponex and 10% biogas wastewater 20 2.2.2.2 The lab experiments 21 2.2.3 Methods of water quality analysis 23 2.2.4 Other methods 24 2.2.4.1 Cultivation method 24 2.2.4.2 Harvesting duckweed method 24 2.2.4.3 Biomass monitoring methods 24 2.2.5 Evaluating software testing techniques based on the standard deviation 28 2.2.6 Model 28 CHAPTER RESULTS AND DISCUSSION 30 3.1 Raw swine wastewater characteristics 30 3.2 Evaluation of Digital Image Analysis (DIA) by using Adobe Photoshop software 31 3.2.1 Determine an optimal subject to be analyzed 31 3.2.2 Determine an optimal distance to analyzed 31 3.3 Comparison between digital image analysis and gravimetric methods 32 3.3.1 Experiment in a 300 ml condition 32 3.3.2 Experiment in a 3.4 L conditions 34 3.4 Specific growth rate of duckweed 35 iii 3.5 Wastewater treatment efficiency of duckweed in flow reactors 37 3.6 Determination of Equal-size mixed flow reactor in series kinetic coefficients 39 CHAPTER CONCLUSIONS AND RECOMMENDATIONS 41 4.1 Conclusions 41 4.2 Recommendations 42 REFERENCES 43 iv LIST OF FIGURES Page Figure 2.1 Lemna Minor was cultivated in CETASD .18 Figure 2.2 Sampling site at a pig farm in Hoai Duc 18 Figure 2.3 Swine wastewater sampling process 19 Figure 2.4 Sampling Duckweed 19 Figure 2.5 Three conditions 20 Figure 2.6 Schematic diagram of the lab-scale continuous flow treatment system with the duckweed 22 Figure 2.7 Lab-scale treatment system with the duckweed 22 Figure 2.8 Duckweed cultivation Procedure 24 Figure 2.9 Step of Lemna Minor harvesting 24 Figure 2.10 Experimental ponds .25 Figure 2.11 Examples of Experimental ponds containing a control patch (in red) 26 Figure 2.12 Calculation of the pixel of red patch 26 Figure 2.13 Calculation of the pixel of leaf 27 Figure 2.14 The pictures of duckweed taken from different distances 28 Figure 3.1 Fluctuation of the surface area and dry weight by Lemna Minor in two bottles of experiment 33 Figure 3.2 Correlation between the digital image analysis and the gravimetric method 34 Figure 3.3 Correlation between dry and fresh weight .35 Figure 3.4 Correlation between the weight density and growth rate of duckweed 36 Figure 3.5 Variation of specific growth rate and the number of reactor 37 Figure 3.6 The relationship between COD; NH4+-N; TN; TP removal curve 38 Figure 3.7 Fluctuation of kinetic coefficients 40 v LIST OF TABLES Page Table 1.1 The Figure for Breeds in Vietnam in five years Table 1.2 The daily mass of swine manure including feces and urine Table 1.3 Properties of pig wastewaters in Singapore farms .6 Table 1.4 Advantages and disadvantages of technologies Table 1.5 Comparison of components between Duckweed and duckweed fern in weight after drying 11 Table 2.1 Name of common lab equipment 17 Table 2.2 The compositional formula of 1000-fold diluted Hyponex 21 Table 2.3 Hydraulic retention time (HRT) of 10 ponds .23 Table 3.1 Characteristics of raw swine wastewater 30 Table 3.2 Standard deviation value for the different access point at each distance 31 Table 3.3 Standard deviation value for different access range .32 Table 3.4 Fluctuation of duckweeds surface area in two conditions 32 Table 3.5 Comparison of kinetic coefficients for COD, TN, TP, NH4+-N for series of the experiment .39 vi LIST OF ABBREVIATIONS ASEAN Association of South East Asian Nations ADAB Australian Development Assistance Bureau BOD Biochemical Oxygen Demand CETASD Center for Environmental Technology and Sustainable Development COD Chemical Oxygen Demand FAO Food and Agriculture Organization GTZ German Agency for Technical Cooperation HRT Hydraulic Retention Time IDRC Canada’s International Development Research Centre MONRE Ministry of Natural Resources and Environment QCVN National Technical Regulation SS Suspended Solid TCVN Vietnam Standard TN Total Nitrogen TP Total Phosphorous TS Total Solid TSS Total Suspended Solids UNDP United Nations Development Programme UV Ultraviolet-visible VNU Vietnam National University vii INTRODUCTION Significant of the study There is no doubt that livestock industry plays an important role in the Vietnamese economy in which, agriculture sector accounts for 15.34% GDP (General Statistical Office, 2017) The livestock industry brings great benefits for the country; however, it creates an undeniable threat of pollution affecting the health of the community and natural ecosystems Clearly, one of the biggest problems is wastewater from farm flowing into receiving resources without being treated or handled by single, effective measures as regulated in the discharge standards Especially swine wastewater which contains pollutants and pathogenic microorganisms, is many times higher than the emission allowed According to General Statistics Office of Vietnam, the total number of pigs in October 2016 was 29.1 million (Thống kê chăn nuôi Việt Nam, n.d.) Pigs accounted for 7.2% of total livestock husbandry, 72% of total meat production It was estimated that in 2016, the flocks of pigs in the whole country discharge 21.2 million tons of manure, 10.6 million tons of urine and about 200 million m3 of wastewater According to the summary report of the Institute of Animal Husbandry (Antoine Pouilieute et al., 2010), most of the livestock farmers leave the wastewater flowing freely into the surrounding environment, which causes stinking odor, especially on hot days The concentration of H2S and NH3 gas is about 30-40 times higher than the permissible level The total number of microorganisms and spores is also several times higher than permitted (Xuan An, 2007) In addition, the concentration in COD, total nitrogen and total phosphorus in animal wastewater are very high Pollution levels tend to increase with the scale of production Several big farms have already applied wastewater treatment technologies, however, in household pig farms this issue has been ignored There are many reasons explaining why such as the social awareness, high cost, shortages in wastewater management and scattered small-scale Consequently, there is an urgent we have used commercial liquid fertilizer for duckweed This type of liquid fertilizer does not contain carbon compounds 3.3.2 Experiment in a 3.4 L conditions The relationships between the leaf area and weight (dry or fresh) are expressed in equations 3.1, 3.2, Figure 3.2 It can be seen that both curves present a correlation coefficient of determination (R2) was higher than 0.8, showing a direct and linear relationship between the methods used to indirectly determine the biomass content and the duckweed growth Therefore, in our test conditions, digital image processing can be used as a satisfactory method to determine the biomass of duckweed ( ) ( ) ( ) (3.1) ( ) ( ) ( ) (3.2) Figure 3.2 Correlation between the digital image analysis and the gravimetric method These results are consistent with findings published by (Tabou et al, 2014) demonstrated image processing method allows for convenient tracking and nondestruction of duckweed parts Although these results are very interesting in determining the growth dynamics of L minor in the laboratory as well as in the field, the small fractional recovery rate L minor will not produce the same weight 34 for the same area The concentration of nutrients in environment influence the growth and the shape of duckweeds (Hillman, 1961) (Caicedo, 2000) 3.4 Specific growth rate of duckweed Figure 3.3 represents the relationship between dry weight and fresh weight of duckweed interpolated by mean of the equation 3.3; three lines 1;2;3 below represent correlative value from three sampling duckweed Figure 3.3 Correlation between dry and fresh weight From the figure 3.3 it can be observed that from the first to the third sampling the duckweed, ratio of dry weight to fresh weight decrease due to the increase of water accumulation Therefore, dry matter in duckweed got less Besides, the dry weight accumulated in the duckweed account for to 5% of fresh weight This result is similar to the previous research (Timmerman, 2016) found dry matter content in duckweed ranges from to 14% fresh weight corresponding to the development cycle 35 According to the equation showing the correlation between surface area and biomass (FW; DW) in section 3.3.2, we can determine DW and FW from the surface area of duckweed at the specific sampling time Moreover, the correlation between weight density and growth rate can be described as equation 3.3: ( ) ( ) ( ) (3.3) Figure 3.4 Correlation between the weight density and growth rate of duckweed This relationship also represented in Figure 3.4 The slope of the straight line corresponds to the specific growth rate (SGR), a basic parameter for biomass determination The average value of specific growth rate during the experiment period (5 days) was calculated in dry weight and fresh weight about 0.008625 and 0.008649 , respectively These results are consistent with the found results of other comparative studies of duckweed in comparable similarity, suggesting that SGR does not change substantially at genus or species level, but reflects adaptation of individual cell to specific local conditions (Ziegler, 2015) From the experiment system described in section 2.2.2.2 each reactor will have a respective SGR value Applying equation 3.3, coefficient (or SGR) in reactors will be calculated and described in the following chart of Figure 3.5 The black, red, blue lines respectively illustrate results from three times sampling 36 Figure 3.5 Variation of specific growth rate and the number of reactor In general, the trend of specific growth rate from 1st to 3rd reactor is increase Then, it dropped until 5th reactor It can be explained on the first day that the concentration of pollutants in wastewater is higher, in that environment, duckweed must adapt to growth The rate of this day is lowest On the last day, the concentration of nutrients decreases after removing by duckweed The result of SGR corresponds to dry weight with greater dispersion than fresh weight due to the change in dry matter content during the development of duckweed The fluctuations of the growth rate from the sampling correspond to a decrease in concentrations of pollutants in wastewater The difference is caused by the weather factors 3.5 Wastewater treatment efficiency of duckweed in flow reactors Figure 3.6 illustrates the variation in COD, NH4+, TN, TP according to the hydraulic retention time (HRT) between duckweed system and witness The red line presents the change of concentration in self-treatment system The blue line shows the evolution of parameters in duckweed system 37 Figure 3.6 The relationship between COD; NH4+-N; TN; TP removal curve in two systems Depending on the initial concentrations, Duckweed treatment system (DTS) removed between 35 and 59 % of the initial COD; between 38 and 68 % of the initial N-NH4+; 17 and 39% of total phosphorus; 13 and 57% of the total nitrogen in days (Figure 3.6) In general, nutrients removal efficiencies were found to be comparable to other studies on the system (Alaerts G J., 1996) (Körner, 1998), (Mohedano, 2012) expressed that using L gibba removal 34–99% for TN and 14– 99% for TP According to (Zimmo, 2004) the process of removing nitrogen can occur in many ways: sedimentation, volatilization, nitrification and denitrification, removal by duckweed and microorganisms Compared to self-treatment systems, the results indicate that natural processes including physical processes can reduce levels of pollutants In addition, bacterial involvement is a significant factor in this 38 process Additionally, some of the factors which may affect the treatment process are as different hydraulic retention times, water depths, initial nutrient concentrations, duckweed densities and harvesting regimes 3.6 Determination of Equal-size mixed flow reactor in series kinetic coefficients A first-order model was used to describe the COD, TN,TP and NH4+-N reduction in the series reactors with the first-order rate constants being calculated from a linear regression using equation 3.4 Based on this calculation, the first-order rate constants of COD, TN, TP and NH4+-N removal are shown in Table 4.5 Table 3.5 Comparison of kinetic coefficients for COD, TN, TP, NH4+-N for series of the experiment HRT (day) kCOD (day-1) Standard error kTN (day-1) Standard error k NH4+-N Standard kTP Standard -1 -1 (day ) error (day ) error 0.089 0.012 0.079 0.009 0.084 0.013 0.084 0.013 0.062 0.011 0.057 0.008 0.071 0.014 0.071 0.014 0.043 0.007 0.067 0.017 0.067 0.014 0.067 0.014 0.036 0.005 0.054 0.011 0.057 0.011 0.058 0.012 0.030 0.005 0.036 0.007 0.037 0.007 0.036 0.040 Nutrient removal coefficients (k, Table 3.5) were consistent with results from the previous systems using the same initial nutrient concentrations The values for COD, TN, TP, NH4+, were in the range of 0.03-0.089; 0.036-0.079; 0.036-0.084; 0.037-0.084 (per day) (Benjawan, 2008) found removal coefficients were in the range of 0.048-0.074 and 0.047-0.0780 in duckweed systems, for TN and NH4+ respectively with similar nutrient concentrations applying for domestic wastewater system These values were in the same range as the first-order rate constants found 39 for the change of nutrients using floating aquatic macrophytes (Sooknah, 2004) These kinetic coefficients can be useful in forecasting the removal rate or system efficiencies in system designs duckweed From the experiment system described in section 2.2.6 each reactor will have a respective k value Figure 3.7 display the fluctuation of kinetic coefficients The black, red, blue lines respectively illustrate results from three times sampling and the pink line represents the average value Figure 3.7 Fluctuation of kinetic coefficients In general, the trend of from 1st to 5th reactor is decrease However, the second sampling result tended to be different due to the influence of weather factors during the test such as rainfall Compared with the average value of data obtained with the dispersion is quite large by experiments done in natural conditions are affected by multiple factors such as weather, temperature and so on 40 CHAPTER CONCLUSIONS AND RECOMMENDATIONS 4.1 Conclusions The present research has evaluated the ability of digital image analysis method to monitoring the growth of duckweed; found the relationship between biomass weight and surface area; calculated the specific growth rate by using this method; and determined the kinetic coefficients for nutrients removal The main findings in this research can be highlighted as follows: (1) Digital image analysis is a newly developed method, application of DIAM can save much time and labor for collection of biomass, making lab determination of biomass, can be applied in lab pond system, and later may be used in the field It can be started from any given point in the photo at the same distance The distance from the surface of the camera to the duckweed must be as short as possible to ensure the accuracy of reasonable quality data Digital image analysis processing by using Adobe Photoshop can be used to show the growth of duckweed (2) Relationship between area measured by DIAM and biomass weight (fresh and dry weight) show by these equations: ( ) ( ) ( ( ( ) ) ( ) ) ( )( ( )( ) ) The dry weight accumulated in the duckweed account for to 5% of fresh weight (3) Average duckweed specific growth rate during the experiment period (5 days) was calculated in dry and fresh weight respectively about 0.008625; 0.008649 ( ) 41 (4) Removal efficiency of duckweed in lab ponds varied between 35 and 59 % of the initial COD; between 38 and 68 % of the initial N-NH4+; between 17 and 39% of total phosphorus; and between 13 and 57% of the total nitrogen in days (5) The kinetic coefficients of COD, TN, TP, NH4+ removal by L Minor were determined, they were in the range of 0.030-0.089; 0.036-0.079; 0.0360.084; 0.037-0.084 (per day) 4.2 Recommendations In the future, we are planning to explore the following contents: (1) Hydraulic retention time and initial concentration play an important role in the nutrients removal of duckweed Thus, the relationship between removal rates along with the length of experiment period and higher initial concentrations should be studied further (2) The concentration of pathogens is one of the important indicator for evaluating the quality of wastewater treatment systems Therefore, the ability to remove pathogens, as well as some specific chemicals such as antibiotics, of duckweed will be taken into consideration in the future (3) Based on the existing of data at the laboratory scale, the next objective is expanding the experimental system to pilot to consider the possibility of application in practice 42 REFERENCES Vietnamese Antoine Pouilieute, Bùi Bá Bổng, Cao Đức Phát (2010) “Chăn nuôi Việt Nam triển vọng 2010” Ấn phẩm tổ chức PRISE Pháp Bùi Xuân An (2007) “Nguy tác động đến môi trường trạng quản lý chất thải chăn nuôi vùng Đông Nam Bộ” Đại học Nông Lâm Thành phố Hồ Chí Minh Nguyễn Thị Thùy Dung nkk (2015) “Đề xuất số giải phát bảo vệ môi trường cho quy trình chăn ni lợn trang trại chăn nuôi địa bàn Huyện Gia Lâm, Hà Nội” Tạp chí khoa học phát triển, 13(3), 427-436 Bùi Hữu Đồn, Nguyễn Xn Trạch, Vũ Đình Tơn (2011), “Quản lý chất thải chăn nuôi”, Nhà xuất Nông nghiệp, Hà Nội Cao Thế Hà nkk (2015) “Vai trị cơng tác đánh giá chất lượng nước thải chăn nuôi lợn việc xác đinh công nghệ xử lý” Tạp chí khoa học cơng nghệ Việt Nam, Tập , Số , tr 50-55 Đặng Đình Kim nkk (2005) Nghiên cứu tảo độc nước Việt Nam Báo cáo hội nghị tồn quốc ni trồng thủy sản Hải Phịng Hồng Thị Như Phương nkk (2015).“Bèo tấm, lời giải tiềm cho toán bùng nổ dân số tồn cầu” Tạp chí Khoa học Công nghệ Việt Nam, Tập 2, Số 6, tr 28-36 Tổng cục thống kê (2012-2016) “Niên giám thống kê” Nhà xuất thống kê Hà Nội English Alaerts G J (1996) "Performance analysis of a full-scale duckweed-covered sewage lagoon" Water Research, Vol 30, Iss 4, pp 843-852 Bekcan S., Atar H H., Beyaz A (2009) "Measurement of the Effects of Liquid Fertilizers at the Different Levels on Duckweed (Lemna Minor L.) 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UNIVERSITY DINH THI TO UYEN DIGITAL IMAGE ANALYSIS OF DUCKWEED GROWTH IN SWINE WASTEWATER AFTER ANAEROBIC TREATMENT MAJOR: ENVIRONMENTAL ENGINEERING RESEARCH SUPERVISOR: Assoc Prof Cao The Ha Prof Jun... and growth rate  Evaluation of the efficiency of duckweed in swine wastewater treatment  Practical application of digital image analysis to monitoring nutrients removal of duckweed Scope of