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

VIETNAM NATIONAL UNIVERSITY, HANOI VIETNAM JAPAN UNIVERSITY DINH THI TO UYEN DIGITAL IMAGE ANALYSIS OF DUCKWEED GROWTH IN SWINE WASTEWATER AFTER ANAEROBIC TREATMENT MASTER'S THESIS

VIETNAM NATIONAL 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

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

TABLE OF CONTENTS

ACKNOWLEDGMENTS i

TABLE OF CONTENTS ii

LIST OF FIGURES v

LIST OF TABLES vi

LIST OF ABBREVIATIONS vii

INTRODUCTION 1

LITERATURE REVIEW 4

CHAPTER 1 Swine wastewater and environmental impacts in Vietnam 4

1.1 1.1.1 The status of livestock development in Vietnam 4

1.1.2 The status of swine wastewater after anaerobic treatment in Vietnam 5

1.1.3 Environmental impacts of animal waste 6

Swine wastewater treatment technologies in the world and Vietnam 8

1.2 1.2.1 Popular treatment technologies 8

1.2.2 Phytoremediation 9

Duckweed as a potential plant for phytoremediation of swine wastewater 1.3 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

Growth evaluation using Digital Image Analysis processing 14

1.4 1.4.1 Popular methods to evaluate plant growth 14

1.4.2 Assessing duckweed growth by measuring frond area with software 15

MATERIALS AND METHODS 17

CHAPTER 2 Materials and instruments 17 2.1

2.1.1 Chemicals and instruments 17

2.1.2 Duckweed 17

2.1.3 Wastewater 18

Methods 19

2.2 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

RESULTS AND DISCUSSION 30

CHAPTER 3 Raw swine wastewater characteristics 30

3.1 Evaluation of Digital Image Analysis (DIA) by using Adobe Photoshop 3.2 software 31

3.2.1 Determine an optimal subject to be analyzed 31

3.2.2 Determine an optimal distance to analyzed 31

Comparison between digital image analysis and gravimetric methods 32

3.3 3.3.1 Experiment in a 300 ml condition 32

3.3.2 Experiment in a 3.4 L conditions 34

Specific growth rate of duckweed 35 3.4

Wastewater treatment efficiency of duckweed in flow reactors 373.5.

Determination of Equal-size mixed flow reactor in series kinetic coefficients 393.6

CONCLUSIONS AND RECOMMENDATIONS 41CHAPTER 4

Conclusions 414.1

Recommendations 424.2

REFERENCES 43

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

LIST OF TABLES

Page

Table 1.1 The Figure for Breeds in Vietnam in five years 4

Table 1.2 The daily mass of swine manure including feces and urine 5

Table 1.3 Properties of pig wastewaters in Singapore farms 6

Table 1.4 Advantages and disadvantages of technologies 8

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 2 series of the experiment 39

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

TSS Total Suspended Solids

UNDP United Nations Development Programme

UV Ultraviolet-visible

VNU Vietnam National University

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

need in the treatment of wastewater generated by the livestock industry It is impossible that a single method can be applied to solve all problems of untreated wastewater It must be a combination of many methods to prevent the secondary pollution and minimize the cost of processing Many techniques have been applied such as anaerobic, anoxic combining with the aerobic process, ecological technology using aquatic plants However, due to the characteristics of swine wastewater like organic components, suspended solids, nutrients and so on make it difficult for wastewater treatment system operation, and significantly reduce the treatment capacity It is crucial to recovering and removal nutrients in swine wastewater before discharging to the drainage system This not only reduces the load of contaminated wastewater but also increases the system efficiency, as well as take advantages of the resource for other multiple purposes

For all the reasons mentioned above, we suggested studying on duckweed as a potential, eco-friendly phytoremediation in pig farm wastewater treatment This research aims to apply digital image analysis software in the measurement of duckweed surface area and growth rate to monitor nutrients absorption of duckweed,

we decide to study on “Digital image analysis of duckweed growth in swine

wastewater after anaerobic treatment.”

Objectives of the study

 Applicability of digital image analysis software in the measurement of

duckweed surface area 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 the study

This thesis presents the results of research on a biological treatment technology using duckweed with laboratory scale experiments, which is suitable for

pollutants removal and effluent reuse of 10-fold diluted real swine wastewater Special attention was paid to target parameters including chemical oxygen demand (COD), Total Nitrogen, Total phosphorus, Ammonium (NH4+) during the period of experiment from Jan to June 2018

Thesis’s outline

This thesis combined and displayed the results from study which was conducted during master program at Environmental Engineering Program, Vietnam Japan University

Chapter 1: Literature review gives information on the status of swine

wastewater after anaerobic treatment in Vietnam focusing on technologies the potential of duckweed as a phytoremediation as well as methods to evaluate plant growth

Chapter 2: Materials and methods describes methods to evaluate applicability

of digital image analysis software in the measurement of duckweed surface area, the growth rate and the efficiency of duckweed in swine wastewater treatment Finally, the practical application of digital image analysis to monitoring nutrient absorption

of duckweed were analyzed

Chapter 3: Results detail and discussion focus on raw characteristics of swine

wastewater; evaluation the ability of digital image analysis to showing the relationship of duckweed compared with gravimetric methods; kinetic modeling

Chapter 4: The conclusion summaries the main finding, implication and

limitation of finding as well as a suggestion for future research

LITERATURE REVIEW CHAPTER 1.

Swine wastewater and environmental impacts in Vietnam

1.1.

1.1.1 The status of livestock development in Vietnam

In recent years, the domestic animal breeding industry has always maintained

a high and stable growth rate, contributing to the overall growth of the agricultural sector According to statistics, the annual number of pig and poultry the trend is increasing, the number of buffaloes and cows is stable Among livestock, pig production is common and in terms of production, pork contributes about two-thirds

of the market demand The table 2.1 below shows the number of livestock in the period from the year 2012 - 2016

Table 1.1.The Figure for Breeds in Vietnam in five years

Units: Million heads

2015, there are about 29,000 breeding farms (General Statistics Office, 2015) By

2016, the number of farms has increased to over 33,000 farms

1.1.2 The status of swine wastewater after anaerobic treatment in Vietnam

When animal husbandry is concentrated, the density of livestock increases, resulting in high levels of pollutant load and pollutants concentration According to the research of DLP-MARD in 2015, pig farming generated the highest proportion

of manure (30.3 percent) Pig farming was concentrated mainly in lowlands and populated areas It causes the greatest pollution compared to other animal farming species (Dinh, 2017) Pig manure is also in slurry form and not easily collected Besides, it has discharged the highest percentage of manure directly into the environment (DPL-MARD, 2015a) The daily mass of feces and urine excreted by a pig that emits about 6-8% of its mass To produce 1000 kg of pork, 84 kg of urine,

39 kg of fertilizer, 11 kg of TS, 3.1 kg of BOD5, 0.24 kg of N-NH+4 were added per day (Bùi Hữu Đoàn và nkk, 2011) So, concentrated animal husbandry is one of the major sources of high-level waste that pollute the environment in our country

Table 1.2 The daily mass of swine manure including feces and urine

waste and wastewater should be separated or not), specific conditions of each province Information on swine wastewater has been adequately covered by a large international project coordinated by many prestigious agencies such as UNDP, FAO Data are presented in table 1.3

Table 1.3 Properties of pig wastewaters in Singapore farms

Parameter Average concentration

in each country These pollution values are not up to the standards of the sector of livestock wastewater treatment 10 TCN 678:2006 and exceeds several times the stringent standards of National Technical Regulation on the effluent of livestock QCVN 62-MT:2016/BTNMT (MONRE, 2016)

1.1.3 Environmental impacts of animal waste

Manure and wastewater from livestock not only pollute the air but also contaminate soil, water surface and groundwater The decomposition of organic compounds in animal manure (especially protein in anaerobic conditions) often produces odorous gases (hydrogen sulfide, ammonia and so on) causeing air pollution The livestock wastewater, which contains high N, P contents, without proper treatment may cause eutrophication of the receiving water bodies, leading to the phenomenon of "algae bloom", ecological imbalance and deterioration of water

quality (Đặng Đình Kim và nkk, 2005) When animal husbandry is concentrated, densities of livestock will be intensified This results in high levels of pollutant load and concentration, which adversely affect the environment and public health (Vu

Thi Nguyet et al., 2014)

Livestock waste affects the environment and human health in many ways, such as polluting surface water, groundwater, air, soil, and agricultural products This is the cause of many respiratory and digestive diseases because the waste contains many pathogens The World Health Organization has warned that without adequate measures to collect and treat animal waste, it will have a huge impact on human health, livestock and environment Particularly, viruses that mutate from diseases such as foot-and-mouth disease, blue ear pig disease can spread rapidly and can kill many people (WHO, 2001)

Currently, no public assessment report and full details about the environmental pollution caused by the livestock sector According to the final report of the National Research Institute of Animal Sciences, most of the livestock wastewater are used for direct irrigation of crops, fish culture or discharge to the surrounding environment, causing stinky odor, especially on hot days The concentration of H2S and NH3 is about 30-40 times higher than the permitted levels In normal conditions,

H2S is one of the causes of odor problems The total number of microorganisms and spores is higher than allowed level (An, 2007)

Livestock waste must be controlled by managers, community and stakeholders, which can collaborate to reach consensus from many aspects, such as limiting environmental pollution, protecting human health, residential landscapes as well as

no inhibiting the development of the sector

Swine wastewater treatment technologies in the world and Vietnam

1.2.

1.2.1 Popular treatment technologies

Currently, there are many swine wastewater waste treatment technologies applied in Vietnam including manure composting, biogas technology, biological agents, bio-ponds, etc

Processing method pig traditionally the most popular from production systems detention is the lagoon anaerobic, with the application of land next 5 substance floating on the ground and mud to use nutrients and eliminate sources discharge points This method has been proven to be the most economical method of waste disposal because there is no aeration is needed, but unfortunately, it is the cause of most of the odors related to pig waste The production of many odorous compounds because of bio-fermentation becomes more concentrated during anaerobic digestion (Miner, 1995)

Currently, there are many livestock waste treatment technologies applied in Vietnam including manure composting, biogas technology, biological agents, bio-ponds, etc Table 1.4 below lists advantages and disadvantages of the different technologies

Table 1.4 Advantages and disadvantages of technologies

Wastewater stabilization ponds system

Low cost Produce effluent well-suited to

irrigation High removal efficiency (80%)

Modification flexibility

Large area Depending on climate Odor emission The risk of soil erosion

Anaerobic filter tank system

Durable structure

Small area Less time in construction

Difficult to maintain the system,

monitoring Annual bottom sludge dredging is required

No flexibility Secondary treatment required

Biogas capture system: fixed dome, covered lagoon, channel digester

Eco-friendly Low cost Reduce soil and water pollution

Generate biofuel

(Homebiogas, n.d.)

The systems used in the production

are not efficient Impurities Depending on climate Suitable for rural area

1.2.2 Phytoremediation

Natural treatment systems using aquatic plants are consist of wastewater stabilization ponds, wetlands (surface, subsurface, and vertical flow), and floating aquatic plants (duckweed or hyacinth)

The main advantage of these systems is that they consume less power, reduce operation time and maintenance, and lead to lower construction and operation costs than systems that use machines The major disadvantage is that the input wastewater control must be more serious as the treatment depends on the climatic factors and the large areas of land required (Crites, 1998)

Phytoremediation is the absorption of pollutants in water by species It is the use of plants to attract, concentrate and accumulate pollutants from the aquatic environment into the roots, leaves, body, buds and so on These contaminants are then removed from the environment through plants harvested This method is used very effectively in many wetlands with organic pollutants such as heavy metal As,

Pb, etc Some of the common species used in this method are water hyacinth, duckweed

Duckweed as a potential plant for phytoremediation of swine wastewater 1.3.

treatment after biogas

1.3.1 General characteristics of duckweed

Duckweed, a member of the Lemnaceae family, is a group of aquatic plant

distributed throughout the world, particularly in the tropics and subtropics It is an important food source for some aquatic organisms, such as waterbirds, fish and

poultry (FAO, 1999) Currently, Lemnoideae has 37 species belonging to 5 genera:

Spirodela, Landoltia, Lemna, Wolffiella, and Wolffia (Hoàng Thị Như Phương và

nkk (2015)) In water-based ecosystems, duckweed is a group of organisms that play a major role in overcoming eutrophication in freshwater bodies as they can grow rapidly and steadily and taking up most of the nutrients substances At the same time, this group of aquatic plants is also used for assessing the environmental

quality of water, bring many valuable achievements of science and practice (Cao et

al., 2015)

Duckweed is distributed worldwide but is common and most diverse in the tropical regions and subtropics In temperate regions, they can develop well during the summer It usually appears in areas with standing water or slow runoff Landolt (1986) published research on the geographic distribution of duckweed from more than 30 years ago The adaptability of duckweed is demonstrated by its presence in various climate zones without significant phenotypic differences (Lam E A K., 2014) (E, 1986) Rapid growth is usually observed in small ponds and swamps, as these areas are rich in nutrients Some species also survive in saline water (up to

2.5% NaCl for Lemna minor), but they do not accumulate Na+ ions during growth (FAO, 1999)

Table 1.5 Characteristic of individual duckweed species (Genera) Lemna Spirodela Landoltia Wolffiella Wolffia

Unlimited growth in nutrient-rich water

Forms turions during winter

grows in the shade;

diverse water quality;

Less common

does not indicate the nutrient rich environment

Nutrient-rich still waters with a depth

of a meter

Nitrogen and nutrient-rich water, capable to grow in full sunlight

Shallow, fresh and nutrient-rich waters

(Timmerman M e., 2016)

Lemnaceae has been mentioned because of the amount of protein to be one of

the highest in the plant (Table 1.6) It can reach up to 40% with optimal condition

Table 1.6 Comparison of components between Duckweed and duckweed fern

in weight after drying

The sustainable agriculture is urgent to limit the impact of climate change as well as to provide new sources of livestock And once more, duckweed is considered as a model plant because of its differences from other plants It can be used to treat pollution, fuel generation and biomass production with little or no soil requirement (Lam E A K., 2014)

1.3.2 Factors affecting the growth of duckweed

Duckweed requires solar energy and minerals for its photosynthesis At

optimal conditions (water temperature, pH, light density, nutrition), L minor could

double biomass within 16- 24 hours (FAO, 1999)

According to theoretical calculations, with this growth rate, after only 50 days, duckweed can fully cover 1 hectare with initial amount of 10 cm2 and will cover the entire area of 32 hectares after 60 days (FAO, 1999) Several major factors affect the growth and development of duckweed: (Zhang K, 2014)

Water temperature: from 6-33oC, the preferred temperature is 30oC

 pH: The most suitable pH value is 6.5-7 but can it can survive in the range of

pH 5-9 The pH determines the most frequent form of N and S in the chemical equilibrium of NH4+/NH3 and H2S/S-, which are common products

of the anaerobic process (McLay, 1976)

 The concentration of mineral: duckweed also needs nutrients and minerals to grow, but in environments with high concentrations of minerals, duckweed grows slower than in those with low contents of mineral

 Water depth: In warm weather, the depth of water does not affect the growth and development of duckweed However, this is a big problem in hot or cold climates because the water temperature will fluctuate greatly and will not be kept at the optimum temperature range In addition, the depth of water for duckweed should be calculated

1.3.3 The ability of duckweed in treating pollution

According to Selvarani et al., (2015) determined the efficiency of municipal wastewater, drainage and wastewater treatment of L minor The experiment was

conducted at four different dilutions, such as raw water, 25%, 50% and 75% dilution In each experiment, duckweed was added at a rate of 0.6 kg wet/m2 Water quality parameters were analyzed once a week The removal efficiency of up to 96% NH3-N was observed for the dilution of 25% L minor achieved the maximum

removal efficiencies of 96%, 98%, 98%, 96%, 79% and 79% for NH3, NO2-, NO3-,

PO43-, BOD and COD in municipal wastewater, respectively (Selvarani, 2015)

Nassar et al., (2015), using L minor for reclamation of agricultural wastewater

and produce high protein feed The removal efficiencies of phosphorus and nitrogen from raw wastewater with HRT for 10 days were 76.9% and 68.3%, respectively Meanwhile, the average fresh and dry yields were 745.8 and 108 kg/ha/day, respectively Dry matter values ranged from 5.5 to 7.2 with an average value of 6.1% Protein and phosphorus content of dry matter were respectively 28.1% and 0.83% Therefore, it is possible to supplement duckweed to animal, poultry and fish feed without the expense of rice bran (ISCDRA, 2015)

Another study by Showqi et al (2017) used L minor to treat wastewater for

15 days under natural conditions To evaluate the removal effectiveness of different contaminants, duckweed was also cultured in tap water for control The results showed that the levels of nitrogen (N), phosphorus (P) decreased 93.4 and 99.9%, respectively in wastewater and then these elements are observed with increased concentrations in the plant body (Showqi I, 2017)

There are many advantages of duckweed ponds to treating wastewater This system reduces operating costs, consumes less energy Ducks are harvested conveniently and quickly The surface of the lake is completely covered so the parasite is reduced The competition between algae and duckweed will arise due to diatom covered light and energy to the surface (Steen, 1999) The emergence of

duckweed reduced evapotranspiration compared to the witness (Oron et al., 1985)

Biomass harvested is used for many different purposes

However, some disadvantages need to be considered as follows: duckweed growth is inhibited in temperatures too low (<15oC) or too high (> 35oC) The

removal efficiency is lower than the ponds used algal (Steen et al.,1999; Zimmo et

al., 2002)

Growth evaluation using Digital Image Analysis processing

1.4.

1.4.1 Popular methods to evaluate plant growth

The leaves are an organ of the tree Measuring leaf area is very important for many physiological research and agronomy, such as plant growth, photosynthesis and transpiration Direct and indirect methods can be used to measure leaf area The direct method is the destruction of the structure of the leaf They can produce the most accurate results, but they have the disadvantage of being extremely time-consuming and inconvenient when deployed on a large scale Indirect methods have been developed that are harmless, less expensive, and less time-consuming, but they are not very accurate (Kaur, 2014)

The simplest method to measure the growth of a duckling is to count fronds Most studies of clover and phytotoxicity depend on the amount of frond However, counting fronds is easy deceptively Counting fronds in just a few containers can take quite some time The amount of fertilizer will be proportional to the biomass only if the fronds in the different treatments share the same average measurement (same shape) and the same density The effectiveness of different treatments may deny these assumptions

Weight is a clear measure of plant growth However, the weight measurement

of duckweed can produce several problems Harvesting of duckweed can affect their performance It affects the parts of the leaf The volume of roots will also be calculated during weighing Due to the clear separation that may cause interference results

The fresh or wet weight is measured after soaking it very gently with absorbent paper to remove water After that, their fresh and dry mass is weighed immediately Due to the low content of dill material, the weight after drying can be affected However, these weight methods still produce quite accurate results But this process is quite time consuming

Less time-consuming methods involving image processing based on camera and computer program images to analyze these images have been replaced by all

other techniques for evaluating leaf area (Hargrove & Crossley, 1988; Hagerup et

al., 1990) These methods can calculate both the decayed area and the area that was

been affected by outdoor factors, depending on the computer programs used

Image processing provides solutions to these problems and provides an automated explanation of plant images Although there are a lot of strict requirements for the implementation steps, this method will open the future for the application of digital technology in the analysis of growth in various subjects

1.4.2 Assessing duckweed growth by measuring frond area with software

According to Bekcan et al., (2009), Image analysis processing provides an

opportunity to estimate their biomass yields without any intervention (S Bekcan, 2009) It is possible to improve the image analysis techniques, and this can provide better equation to estimate the weight of duckweed with high accuracy

(Can, 2012) found that Adobe photoshop CS helps reduce the analysis time and gives accurate results on leaf surface area When comparing measurements with this software with other software, the number of pixels is calculated more accurately, and the results are more reliable

One company named LemnaTec-a company in Germany introduced the Lemna Tec Scanalyzer software as an image quality software for duckweed Based

on the images captured, the indicators analyzed included quantity, color, frond area and many other parameters This solution improves data quality and reduces the

cost of implementation and the amount of documentation (LemnaTec GmbH (Sd)) (LemnaTec GmbH (sd))

The change in surface area reflects the growth of duckweed Now, the analysis

of the growth process based on digital image analysis opens a new direction and more variety in the collection and processing of data

MATERIALS AND METHODS CHAPTER 2.

Materials and instruments

2.1.

2.1.1 Chemicals and instruments

All chemicals used must be purely for analysis produced by Merck, Fluka companies

Protective equipment: blouse, active carbon masks, gloves, etc

Table 2.1 Name of common lab equipment

This study investigated Duckweed Lemna Minor, which was harvested from

ponds of the local sewage system in Hoai Duc District, Hanoi and then stored at Environmental Technology Laboratory, Center for Environmental Technology and Sustainable Development (CETASD), Vietnam National University (VNU)

Figure 2.1 Lemna Minor was cultivated in CETASD

2.1.3 Wastewater

Wastewater source: Swine wastewater after biogas system were collected from

a household pig farms from Noi hamlet, Cat Que commune, Hoai Duc district, Hanoi The pig farm has a scale of 50 pigs

Figure 2.2 Sampling site at a pig farm in Hoai Duc

Methods

2.2.

2.2.1 Field survey and sampling

Figure 2.3 Swine wastewater sampling process Wastewater samples for this research were taken from storage ponds after biogas in the household swine wastewater treatment Swine wastewater was stirred before picking Sample water was then filtered by using 1mm mesh sieve to remove the sludge, and so on

Figure 2.4 Sampling Duckweed