extfieldsofstudy: Physics Chemistry

text:
Influence of protein concentrate in the diet on productivity and amino acid composition of cow milk

The article presents experimental data on the use of different levels of protein concentrate supplement in the diets of lactating cows with a high proportion of indigestible protein in the rumen. During the course of the research it was observed that the inclusion of Agro-Matic protein concentrate in the amount of 1.0 and 1.5 kg per head per day in the diet instead of other protein feeds increased the level of indigestible (by-pass) protein by 6.3 % and 9.1 %, respectively. The introduction of the maximum amount of Agro-Matic protein concentrate into the diet has contributed to the change in the amino acid composition of milk. It was established that the yield of milk protein during the period of milking was significantly higher in animals receiving different levels of protein concentrate compared to those of the control group. Feeding lactating cows diets with different levels of protein concentrate did not have a negative impact on biochemical parameters of animal blood. All measured blood of experimental animals were within the physiological norms. The maximum content of essential amino acids in the milk of cows from 3 experimental groups was significantly higher and reached up to 1560.9 mg% versus 1480.17 mg% in the control group.

Introduction
The production of high-quality milk and dairy products is of great importance in the economy of Russian Federation; these products are also included in the list of basic human food and fall under the Doctrine of national food security [1][2][3][4][5][6].
In condition of market economy, the problem of low-quality milk production on farms of agricultural enterprises is one among the most relevant [7][8][9][10][11].
With an increase in the milk productivity of livestock, the impact of the non-digestible fraction of the feed protein has also increased. The predominant share of a high degree of cleavage protein in the rumen of cows comes from the protein of haylage, silage and most of grains (wheat, barley, oats, etc.). The optimal ratio of cleavable to non-cleavable protein in feeds for high-yielding dairy cows during the milking period is from 60-70 % to 30-40 % [8,12,13,15,[16][17][18][19][20]. The use of complete feed and feed additives in the diets of highly productive lactating cows contributes to reimbursing the content of deficient and missing nutrients [1-4, 11, 21-22]. The aim of the current study was to investigate the influence of protein concentrate Agro-Matic supplementation to the diet of dairy cows on productivity of animals and amino acid composition of cow milk.

Materials and methods
The current research started in February 2018 on the farm of Agricultural Production Cooperative 'Plemzavod Maisky' in Vologda region, Russia.
The subjects of the research were heifers in the second half of pregnancy and Ayrshire breed cows of older age in dry period. The animals were selected by method of the pair-analogues considering their origin, sex, age, live weight, milk production in the previous lactation period, milk yield over 8200 kg per year and physiological state.
At the time of the experiment the cows and the heifers were eightmonths pregnant with a live weight of 550 kg and average fatness. They were divided into 3 groups of 15 heads each -1 st group a control group, 2 nd and 3 rd groupsexperimental groups. During the course of the study all animals were kept on the same housing and feeding conditions and were clinically healthy.
The animals in the control group received the standard diet used in the farm, balanced in nutritional value according to the norms of feeding developed by All-Russia Institute of Animal Husbandry (2016) [3] and designed to receive milk yield of 39 kg per 1 day during the milking period.
The composition for the cows in 2 nd and 3 rd group (experimental groups) was based on standard diet with addition of different levels of Agro-Matic protein concentrate of 1.0 and 1.5 kg, respectively and simultaneous reduction of other protein-rich feeds. The content of exchange energy and the level of crude protein in protein concentrate enriched diets was equal to the control group.
During the time of experiment milk quality and productivity of cows were measured 3 times a month, respectively, by the method of control milking. The percentage of milk fat was determined in accordance with GOST 5867-90 Milk and dairy products. Method of determination of fat [23] and Gerber acid method described in GOST R ISO 2446-2011 Milk. Method for determination of fat content [24].
Milk samples were taken from 3 animals from each experimental group, collected in one common container and stored in a refrigerator at + 4 ° C. The gross content of essential and non-essential amino acids in cow's milk was determined in the laboratory 'Evonik Chemistry', LLC. Amino acids in the cow milk were determined on a Foss AMINONIR DS2500 spectrometer in the 3rd month of lactation.
Biochemical parameters of blood samples of cows were taken from 9 animals 2 days after the end of the physiological experiment were determined in 'Vologda regional veterinary laboratory'.
The following parameters were measured in animal blood serum: total proteinby refractometric method using IRV -22 Refractometer with the help of The Reis table; ureaby the Michonne and Arnault method by the reaction with paradimethylaminobenzaldehyde; calciumby spectrometric titration with the murexin indicator; phosphorusby colorimetric method based on the reduction of phosphoric-molybdenum acid.
In the blood samples the following parameters were evaluated: glucoseby colorimetric method, color reaction with orthotoluidine reagent (Hultman method in Hyvarinen Nickel modification); caroteneby calorimetric gasoline extraction.
Processing of biometric data was performed by the methods of mathematical statistics (N.A. Plokhinsky, 1969 and E.K. Merkuryeva, 1970) using modern computer programs (standard package of statistical analysis Microsoft Excel 2007).

Productivity and amino acid composition of cow milk
Increasing the productivity of dairy cattle breeding and quality of the final dairy products in the Russian Federation at lower production costs is set among the priority objectives of the State Program until 2022 in the field of modernization of agriculture and industrial safety under conditions of foreign sanctions [1,2,4,5,6,22].
In the course of current study, milk production was determined during 120 days of lactation from the moment of calving onwards. The following indicators were considered: average daily milk yield and gross milk yield with natural fat content and 4% fat content, mass fraction of milk protein and fat, milk protein and fat yield (table 1). Analysis of the experimental data shows that the supplementation of standard diet with 1.0 and 1.5 kg of Agro-Matic protein concentrate has increased the gross milk yield with 4% fat content by 5.93 % and 9.91 %, respectively, compared to animals from control group. There was a marked tendency of increase of gross yield milk with natural fat content and milk with 4% basic in the experimental groups.
In the 3 rd experimental group gross yield with natural fat content was 4297.5 kg, which was significantly higher than in the control group. It should be noted that the gross yield of milk in the 2 nd experimental group and the 3 rd experimental group was 213.3 kg higher than in the control group and 387.4 kg higher compared to animals receiving sunflower cake in the diet. The amount of milk protein in the experimental groups was increased in comparison with animal analogues from the control group. It should be noted that during the experiment the highest content of fat and protein in milk was in the 2 nd experimental group, with the values of 4.03 % and 3.34 %, respectively. Thus, it became evident that supplementation of 1 kg Agro-Matic protein concentrate to standard diets positively impacts milk productivity of cows and significantly increases the mass fraction of milk protein and fat compared to the control group.
During the reference period, the gross yield of milk fat and protein in the experimental groups of animals receiving Agro-Matic protein concentrate in the diet were significantly higher than in animalsanalogues from the first control group. The highest yield of milk fat was observed in cows of the 3 rd group, which was 10.9 % higher compared to theanimals from the control group. Although at the time of the experiment the values of the mass fraction of fat and protein were generally the highest due to the high milk productivity of cows receiving Agro-Matic protein concentrate in the diet, the maximum content of protein and fat was found in cows from the 3 rd experimental group, which was respectively 10.90 % and 9.91 %higher than in the control group. The biological value of milk protein is based on the content of its specific amino acids. It is known that more than 70 % of overall amino acid amount that enters the body is involved in milk formation. The studies conducted on lactating cows with milk yield of 6-8 thousand kg per annum and milk fat content of 3.4 % raised on diets with a balanced composition of exchange protein revealed the deficit of some essential amino acids such as methionine and leucine [5,13,14,15,20,26]. According to Yu.V. Sizova the amount of amino acids to support life are: lysine -36.9 g/day, methionine -6.9, phenylalanine -10.7, and leucine -24.7 g/day [11,21].
Amino acid composition as evaluated in the course of the current study indicates that the inclusion of protein concentrate in the diet positively affected the content of essential amino acids in milk (table  2). There are few main functions of lysine in the animal organism. In unchanged form lysine is a structural component of organs, tissues, and a part of the muscle fibers. Lysine actively participates in the synthesis of collagen and elastin; it is starter molecule in the process of hematopoiesissynthesis of blood hemoglobin and it also stimulates the development of bone tissue. Lysine provides calcium absorption; it participates in the regulation of synthesis and ratio of nucleic acids. Lysine provides synthesis of some hormones and enzymes. Lysine also has a functionin immune system, process of building antibodies. Lysine optimizes the renewal of epithelial tissues and provides normal wool pigmentation. In regard to vitamin metabolism, lisyne controls the conversion of carotene into vitamin A.
Ruminants are able to cover up the need for cystine and methionine by 30-40 % by in vivo synthesis of protein by the specific microbiota of the rumen. Amino acids methionine, proline, glycine and glutamic acid are constantly present in the rumen in amount of 0.1-1.4 mg per 100 ml of the content. Methionine has an important role in the protein synthesis in cattle and other animals, as it is a starting molecule in the biosynthetic parthways of protein in the body of animals.
Analyzing the data obtained in the current study, it should be noted that the maximum content of methionine in milk was found in animals from the 2nd experimental group and its content was 10.2 mg% higher compared to the control group.
The content of lysine in milk was the highest in the group of animals receiving 1.5 kg Agro-Matic protein concentrate in their diet and it was 29.6 % and 0.8 % higher compared to the control group and 2 nd experimental group, respectively. The output of the total amount of essential amino acids in the milk of cows of the 3rd experimental group was evidently higher -5.8 % higher compared to the animals from the control group.
3.2. Biochemical parameters measured in blood of experimental cows The process of metabolism in the body of cows is a series of complex biochemical transformations of nutrients, which are broken down to monomers during the process of digestion and enter the blood and lymph from the digestive system [2,10,22,27].
Biochemical indicators of the whole serum and blood plasma are one of the main indicators of health and physiological state of animals, which fully reflect normal homeostasis and deviations if any. Biochemical indicators can rate diseases and reflect the level of metabolism in the body of dairy cattle. Any deviations in the metabolism of dairy cattle away from normal values can mainly result in decrease in milk production, poor health status and negative effect on reproductive function in general. The composition of biological body fluids (whole serum and blood plasma) reflects the intensity and direction of metabolic processes that occur in animals [1,4,6,28].
To study the biochemical parameters of the blood of cows from the experimental groups 3 cows were selected from each experimental group (table 3). Blood samples were taken 3 hours after morning feeding. One among the main indicators of the level of carbohydrate metabolism in the body of farm animals is the blood glucose concentration. In animals from the 2 nd experimental group, receiving 1.0 kg of protein concentrate in the diet, high blood glucose levels were observed. They were 0.51 mol/l higher compared to the control group.
The main indicator of protein supply in the body is the total protein content measured in the blood serum. Protein is a building material for cells and tissues of the body and is actively involved in milk formation.
Analyzing the data obtained, it should be noted that the total protein content in the blood serum of the animals from the 3 rd experimental group was slightly changed, but during the time of the experiment a significant increase by 9.1 g/l was observed in cows receiving 1.0 kg Agro-Matic protein concentrate in comparison with the control group.
It should be noted that the level of total protein in the blood serum of the experimental cows receiving 1.5 kg of protein concentrate in the diet, has increased by 4.6 g/l (5.9 %) in comparison with the control group, which in turn is a consequence of increased efficiency of the nitrogen utilization in the biosynthesis of milk protein in the body of lactating cows.
To assess the mineral metabolism in the blood serum of animals the level of calcium and phosphorus should be determined. Calcium ions are involved in the process of blood clotting, contribute to the interaction of actin with myosin in skeletal muscle tissue and to the release of neuronal mediator acetylcholine. The calcium level depends on vitamin D and calcium and phosphorus content in the diet of cows. Being the basic element of phosphoric acid, phosphorus participates in all major metabolic pathways in the body of animals. Reactions of phosphorylation are one among main parts in amino acid metabolism and glycolysis. According to data obtained in the current study, animals of the 3 rd


provenance: AgriculturalAndFoodSciences,2019-2020-Agricultural And Food Sciences-2019.jsonl.gz.gz

text:
Batch Investigation of Biogas Production from Palm Oil Sludge, Bambara Nut Chaff and African Wild Mango at Varying Meteorological Conditions

A custom response design study on the biogas production from blends of Palm oil sludge (POS), Bambara nut (VignaSubterranea) chaff (BNC) and African wild mango (AWM) was carried out. The anaerobic digestion was in the ratio of 3:1 and 2:1 of water to waste depending on the nature of the substrates as follows: System A was 100%POS; B: 100%BNC, C: 100%AWM;D: 50%POS+50%BNC and E: 60% POS+20% BNC+20% AWM. The wastes were charged into 32L capacity metal prototype digesters in a batch for 30 days retention period at an ambient temperature range of 26C 37°C. The cumulative gas yield from the five treatments systems (digesters) were different: the 50%POS+50%BNC had the highest cumulative gas yield (69.5L); followed by 100%BNC system (54.5L); 60%POS+20%BNC+20%AWM system (39.2L); 100%POS system (14.4L) and 100%AWM system (11.7L). 100%BNC system had the highest methane content (88.056%); followed by 60%POS+20%BNC+20%AWM system (88.007%); 100%POS system (83.025%); and 50%POS+50%BNC system (73.055%).The research has shown that 100% BNC had the least lag days (6 days), highest calorific value (25330.24KJ/Kg) and highest methane content (88.056%). African wild mango needs to be co-digested to produce flammable biogas. The TS, VS, BOD and TVC were seen to be consistently reducing throughout the digestion period. Meteorological conditions like solar radiation, air temperature and wind speed had significant effects on ambient temperature, slurry temperature, pH and daily gas yield. Keywords— Batch, Biogas production, Palm Oil Sludge, Bambara Nut Chaff, African Wild Mango, Meteorological Conditions. Abbreviations: TS=Total Solid, VS=Volatile Solid, BOD=Biochemical Oxygen Demand, TVC= Total Viable Count

INTRODUCTION
In this era of industrialization and civilization energy turns out to be one of the bedrocks to achieving national economic status. Hence, the importance of energy cannot be over emphasized as it cuts across virtually every work we do from a little household to the largest cooperation company in the world. Some of the challenging facts facing the energy sector worldwide is the exhaustive nature of the commonly and generally used non-renewable energy sources such as petroleum, and the serious threats (uncontrolled emission of greenhouse gases) the use of such energy sources such as petroleum poses to the environment and mankind in general. Also alarming is the poor waste management strategies in the country; these wastes especially bio-degradable wastes constitute nuisance at the road sides, in our streets and sometimes in our homes. It has also been stated that uncontrolled greenhouse gases (CH4, CO2, CO etc) are emitted during the degradation of organic wastes. Subsequent upon these, there have been so many ameliorating technologies employed to ensuring a reduction of the effects such energy sources as it concerns man and his environment; however, biogas production through anaerobic digestion of organic wastes is one of such technologies. Anaerobic digestion is a multistage biochemical process that can stabilize many different types of organic material to produce methane and carbon dioxide by different groups of micro-organisms under anaerobic condition (Joaquin, 2008). Lo et al., (2010) in their work titled "Modelling biogas production from organic fraction of MSW co-digested with MSWI ashes in anaerobic bioreactors" stated that anaerobic digestion involves four steps which are: hydrolysis, acidogenesis, acetogenesis and methanogenesis. The activities of micro-organisims present at different stages of anaerobic digestion however depends on various parameters such as temperature, pH, concentration of nutrients agitation, pre-treatment of feedstocks, carbon/nitrogen ratio, hydraulic retention time etc. Hence, in order to improve the overall efficiency of anaerobic digestion process, there have been many works on biogas production through anaerobic co-digestion of different wastes. Anaerobic co-digestion is defined as the treatment of a mixture of at least two different substrates with the aim of improving the efficiency of anaerobic digestion processes (Neczaj et al, 2012). Oil palm is a common plant in the Southern part of Nigeria. Specifically the south-eastern part believes that it has some traditional values associated with it. Crude palm oil is one of the major products of palm oil processing plants with palm oil effluent as a by-product. This palm oil effluent is often accompanied by palm oil sludge which contains substantial quantities of solids left-over after the liquid must have drained. However, palm oil sludge are sources of pollution to the environment if discharged untreated, this is due to large oxygen depleting capabilities when present in aquatic systems (Hassan et al., 2013). According to Okwute et al., 2007 from their work titled "The environmental impact of palm oil mill effluent (POME) on some physicochemical parameters and total aerobic bio-load of soil at a dump site in Anyingba, Kogi state Nigeria."studies on POME dump site revealed that the physico-chemical properties of the soil at this dump sites were obviously altered due to its acidic nature. Palm oil sludge are produced and discharged into the environment in millions of tonnes from both milling plants and individual households nationwide, thus, posing a big treat to the environment. Anaerobic digestion is the most suitable method for the treatment of effluents containing high concentration of organic carbon such as POME (Borja et al., 1996a). Projection depicts that 555, 457 and 409 million m 3 of methane gas could be produced under high, low and current status production rates scenario respectively up to 2030, if Nigeria harnesses the energy contained in palm oil mill effluent (Ohimain et al., 2014). Vigna subterranean (Bambara nut) is a local bean plant commonly grown in the Northern part of Nigeria, but consumed in all part of the country. After it is processed the chaff are either used as blend for poultry feeds or they are thrown away, thus, depleting the environmental harmony. Previous work done on the biogas production potential of Bambara nut chaff showed that it had potentials for biogas production. According to Ofoefule et al. (2010) in their work titled "Biogas production from blends of Bambara nut (Vignasubterranea) chaff with some animal and plant wastes" observed that Bambara nut chaff has the potential for biogas production though the expected increased biogas yield and extended flammable time was not achieved by the co-substrates employed (cow dung, swine dung, cassava peel and field grass wastes). However, they proposed a chemical treatment to increase the pH to neutrality to enhance gas production and extend the retention time. African wild mango (Irvingiagabonensis) is a plant which produces seeds rich in fat (around 70% on dry matter basis), traditionally used as a soup thickener in central and western African regions. However, it is obvious that much has not been done as it concerns biogas production from African wild mango. Thus, the major objective of this work is to co-digest these three wastes to: (i) determine their physico-chemical properties and their effects on biogas production as well as waste treatment. (ii) to note the effect of African wild mango as a co-substrate with respect to increased gas production (iii) to determine the optimum mix ratio for optimized gas production. (iv) to have an idea of the level of waste treatment achieved at 30 days retention time.

II. MATERIALS AND METHODS
The study adopted custom response design. Bambara nut shells were collected from Ogige market in Nsukka and also from the university environment. African wild mango was gotten from Ibagwa plantation in Nsukka. Palm oil sludge was collected from a local palm oil processing plant in Ajuona community in Nsukka.

SAMPLE PREPARATION
The African-wild mango was chopped in smaller pieces and soaked in water at 100% level for 2 days. Thereafter, it was sieved to facilitate digestion due to the nature of the waste. The same was done for Bambara nut chaff for effective digestion and on-time gas production. After the soaking period, the feedstocks were then weighed out for charging.

EXPERIMENTAL SET-UP
Metallic model biodigesters (Plate 1) utilized for the study were each of 32.0 L working volume (fabricated locally at the National Centre for Energy Research and Development, University of Nigeria, Nsukka). Materials such as top loading balance (Camry Emperors Capacity 50 kg/110 Ibs), plastic water troughs, graduated transparent plastic buckets for measuring daily gas production, the pHep pocket-sized pH meter (Hanna Instruments), thermometers, pressure gauge, thermoplastic hose pipes, metallic beehive stand and biogas burner fabricated locally for checking gas flammability were used.

III.
EXPERIMENTAL STUDY The fermentation of the blends took place for 30 days at the prevailing ambient mesophillic temperature range of 26 o C to 37°C. The ratio of the water to waste in each charging is as shown in table 1. This was based on the moisture content of the organic wastes at the point of charging the biodigesters. Palm Oil Sludge, Bambara Nut Chaff and African Wild Mango were co-digested to result to the following treatment blends: A (100%POS), B (100%BNC), C (100%AWM), D (50%POS+50%BNC) and E (60%POS+20%BNC+20%AWM). Table 1 shows details of the blending. Co-digestion is used to increase methane production from low-yielding or difficult to digest materials. Volume of gas produced, ambient and slurry temperatures, relative humidity and wind speed, insolation, pH and slurry pressure were monitored on daily basis throughout the period of digestion. Flammability check was also carried out on daily basis until the system produced flammable biogas and occasionally till the end of digestion period. The study was carried out at the exhibition ground of National Centre for Energy Research and Development, University of Nigeria, Nsukka.

DETERMINATION OF PHYSICO-CHEMICAL PROPERTIES
The methods used in this work to determine the physicochemical properties of the undigested substrates are clearly defined as follows: The Meynell (1982) method was used to determine the: Total solids and Volatile solids while the A.O.A.C method (1990) was used to determine the: Moisture content, Ash content and Crude fibre content. The Pearson (1976) method was used in the determination of the Crude fat content with the use of Soxhlet extraction apparatus. The Micro-Kjedahl method as described in Pearson (1976) was used in the determination of Crude protein content while the method of surface viable count was used in the determination of the Total viable count (Number of living micro-organisms). The Energy content was determined with bomb calorimeter (model XRY-1A, make: Shanghai Changji, China), using A.O.A.C (1990) method).Walkey- Black (1934) method was used to determine the Carbon content while the ambient and slurry temperature was taken daily using a liquid in glass thermometer and the pH was ascertained using the Hanna instrument pH meter standardized us ing buffer solutions for pH 7.0.The pressure of the gas produced in the biogas digesters was measured daily using the sphygmomanometer. This water displacement method was used to determine the biogas volume while the Bacharach (PCA2) gas analyzer was used to determine the gas composition. A locally made gas burner was used to carry out the gas flammability tests. The population of the microbes in each of the treatment cases was determined at different times (at charging, flammable, peak of production and end of digestion), during the period of study to monitor the growth of the microbes at the various stages.

Data Analysis
The data obtained for each of the systems were subjected to analysis using Microsoft Excel XP 2007. Meteorological data were obtained from Centre for Basic Space Science, University of Nigeria, Nsukka. Table 2 shows the physicochemical properties of undigested wastes.  Table 3 shows the gas compositions for the various substrates.

PROXIMATE ANALYSIS OF THE SYSTEMS
The proximate composition includes the ash, moisture, crude fibre and crude fat contents of the wastes. From table 2, The ash content of the 50%POS+50%BNC system was highest (6%) while that of 100%POS was least (3.9%).The 100%BNC system had the highest crude fibre content (5.4% ) while the 100%POS had the least crude fibre content (2.89%). Each of the wastes for the systems had optimum moisture content because of the mix. Biological activities are increased when digester fluid are mixed to provide homogenous temperature and nutrient condition throughout the digester (Lay et al., 1997). The crude fat for each of the wastes was appreciable.

EFFECT OF C/N RATIO ON THE SYSTEMS
From the results of Table 2 and figure 1, the C/N ratio of (100%POS), (100%BNC), (50%POS+50%BNC) and (60%POS+20%BNC+20%AWM) CD, were seen to be within the range of the optimum C/N. Consequently, each of these digesters flamed. Digesters 100% AWM had low C/N ratio that possibly led to ammonia accumulation and consequently could not flame. C/N ratio is an important indicator for controlling biological systems. During anaerobic digestion, microorganisms utilize carbon 25 to 30 times faster than nitrogen (Yadvika et al., 2004). To meet these requirements, microbes need 20 to 30:1 ratio of C to N.

CALORIFIC VALUES OF SUBSTRATES
The energy contents (figure 2) show that palm oil sludge and bambara nut chaff are good feedstock for biogas production if properly utilized while African wild mango has to be co-digested to produce biogas. Bambara nut chaff had the highest calorific value (25330.24 KJ/Kg); followed by African wild mango (21053.77JJ/Kg) and then Palm oil sludge (16935.98KJ/ Kg).

EFFECT OF WEEKLY BOD, TOTAL VIABLE COUNT, TOTAL SOLIDS, VOLATILE SOLIDS; AND ANAEROB IC DIGESTION AS A WASTE MANAGEMENT TECHNOLOGY
Biochemical Oxygen Demand (BOD) is the amount of dissolved oxygen needed (i.e. demanded) by aerobic biological organisms to breakdown organic materials present in a given water sample at certain temperature over a specific time period. This is a quantitative expression of the ability of microbes to deplete the oxygen in waste water. It is also the amount of oxygen required for the biological decomposition of organic matter in wastewater by bacteria under aerobic conditions. This depletion is caused by the microbes consuming organic matter in the water via aerobic respiration. Total Viable Count (TVC) gives a quantitative idea about the presence of microorganisms such as bacteria, yeast and mould in a sample. The count actually represents the number of colony forming units (cfu) per gram (or per ml) of the sample.Total solid shows the total solid matter constituent of the entire organic waste both degradable and non-degradable. The volatile solid is the true organic matter available for bacterial action during digestion. Figures 3, 4, 5 and 6 show reductions in weekly BOD, TVC, TS, VS respectively. This is expected as the wastes stabilized. Anaerobic digestion is the most important method for th e treatment of food waste because of its techno-economic viability and environmental sustainability. The use of anaerobic digestion technology generates biogas and preserves the nutrients which are recycled back to the agricultural land in the form of slurry or solid fertilizer. The relevance of biogas technology lies in the fact that it makes the best possible utilization of food wastes as a renewable source of clean energy since there is always reduction in BOD, TVC, TS and VS.

THE EFFECTS OF SOLAR RADIATION, WIND SPEED AND AIR TEMPERATURE ON AMBIENT TEMPERATURE, SLURRY TEMPERATURE, PH AND DAILY BIOGAS YIELD
There was variation in solar radiation resulting to highest solar radiation (582.604W/m) on the 26 th day and least (135.655W/m) on the 25 th day. Air temperature had highest (30.309 o C) on the 24 h day and least (23.196 o C) on the 25 h day. Wind speed had highest (1.813m/s) on the 21st day and least (1.035m/s) on the 6th day (figures 7, 8 and 9). The variation in these climatic conditions gave rise to variations in ambient temperature, slurry temperature, pH, and daily volume of gas produced (figures 10, 11, 12, 13 and 14).

V.
CONCLUSION This study has shown that wastes such as palm oil sludge, bambara nut chaff and African wild mangowhich have been termed nuisance to the environment can be utilized to produce biogas which can be used as an alternative to the widely known and used fossil fuel. The digestate after biogas has been produced can also be used as fertilizer to improve plant growth and enhance soil capability in producing. From the results, it can be seen that the palm oil sludge and bambara nut chaff substrates are excellent in producing flammable biogas; capable of being utilized for any purpose such as cooking. African wild mango if co-digested can produce flammable biogas. The research has shown that 100% BNC had the least lag days, highest calorific value and highest methane content.


extfieldsofstudy: Chemistry Physics

text:
Extraction of nitrogen, phosphorus and potassium from food waste under elevated temperature by heat induced bath method

38,000 tonnes of waste is generated per day in Malaysia and 15,000 tonnes of that are food wastes. This waste should be recovered to produce a new valuable product that brings benefits such as fertiliser because it contains various nutrients such as proteins, carbohydrates. This study aims to determine the effects of thermal treatment on the macronutrient of food waste that collected from cafeteria of Universiti Sains Malaysia (USM). Hot water bath was employed as thermal treatment of food waste with five different temperatures (60 °C to 100 °C) with two reaction time (30 min and 60 min). The results have demonstrated increases in the percentage of nitrogen (N) and concentration of phosphorous (P) however for the concentration of potassium (K) shows inversely related after subjected to hot water bath. Thermal treatment showed the untreated sample only consists 2.13 % of N and 31.53 mg/L of P, and after the treatment resulted in an increase in N and P by 0.22 % - 3.39 % and 0.33 mg/L - 21.40 mg/L respectively. On the other hand, the concentration of K in the untreated sample is 0.4915 ppm and after treatment it decreases to 0.1051 ppm - 0.2052 ppm. Based on the range of macronutrient in organic fertilisers in Malaysia, only N was in the range and can directly employ as organic fertiliser while for P and K should undergo further treatment.

Introduction
Food wastes are any food that is not consumed or discarded and generated at any level within food chain where from production, processing, distribution until consumption of food [1]. It biodegradable and the degradation of each component of food waste is affected by environmental conditions, therefore, these wastes need to dispose of properly to provide a clean and healthy environment. Food waste contains various nutrients such as natural fibers, soluble sugars, starches, fats, lipids, proteins, celluloses, and vitamins [2]. There are multi-stages of reaction associated with the production of valuable product (fertiliser and biogas), the first step is crucial and assist in increasing the production of valuable product. Therefore, pretreatment was employed because it can ensure the effective performance of further treatment. Pretreatment is a process where it produced a disrupted, solubilisation and separation of one or more components of biomass [3,4]. It converts complex macromolecules into low molecular weight compounds [5].
Pretreatment could be categorized into physical, physicochemical, chemical, biological and combinatorial pretreatment. Thermal pretreatment is one of treatment where it involves from low to high temperature in the reaction with organic materials. The effect of thermal pretreatment are disintegration of cell membranes of food waste and resulting in the solubilisation [5].
In theory, cell lysis will performed firstly then followed by reducing the size of organic matter molecules [6].
In hot water bath, heat transfer occurs through convection then it followed by conduction. Hot water is the medium of heat transfer because it direct contact with the base of the glassware [7].
Fertiliser is any material used to increase nutrient content to the soil to promote soil fertility and increase the growth of plant. It could improve the crop yield by 50 % to 80 %. Nitrogen (N), Phosphorous (P) and Potassium (K) are the primary macronutrient in the fertiliser.

Waste Collection
Food waste that used in this study was collected from Baktisiswa Cafeteria at Universiti Sains Malaysia. The organic and inorganic waste was separated and only the organic waste was used in this treatment. The segregation of waste were carried to ensure that the waste could be degradable during treatment [8]

Waste Preparation
Food waste was dried using oven dry at 105 o C for 24 hours to eliminate the water content then dry food waste were blended in the heavy duty blender. This is because to increase the surface area to provide better contact time thus enhances the process to breakdown the food wastes during the thermal treatment and to homogenize the composition of the food waste.

Thermal pretreatment
The powder samples were placed in a conical flask with distilled water with ratio 1:10 (W:V) where 25 g of sample with 250 ml of water [9].
Five temperatures were set up which were 60 o C, 70 o C, 80 o C, 90 o C and 100 o C and two reaction times, 30 min and 60 min [6,7]. For the control samples, food waste was placed in conical flask with distilled water at room temperature.

Macronutrient
N, P and K are macronutrients and valuable for plant nutrient. For this research, these macronutrients were analysed before and after thermal treatment 2.4.1. Nitrogen. The method that been used to analyse nitrogen was Total Khejadal Nitrogen (TKN) method [10,11]. TKN procedure basically divided into three parts which were digestion, distillation and titration.

Phosphorous.
For phosphorous analysis, DR2800 spectrophotometer (Hach) was used [11]. The digestion of food waste was carried out first by using microwave digestion method using microwave digester (MARS 6).

Potassium.
Potassium of the food waste were determined by using Flame Atomic Absorption Spectroscopy (FAAS).

Data analysis
The data that obtained be calculated and illustrate as graph by using Microsoft Excel. IBM SPSS is used to analyse the data by two-way ANOVA. This to identify if there is an interaction between the two independent variables on the dependent variable.

Results and discussion
3.1 Effect of thermal pretreatment on macronutrient of food waste 3.1.1 Nitrogen. N in food waste could be found in food which high in proteins such as meat, chicken, fish, and egg. The percentage of N that can be used as organic fertiliser was in the range of 0.7 % -4.4 % [12] In general, as the temperature increases and longer reaction time, the percentage of N will increase. and decreased back at 100 o C.Basically, most N is either tightly bound within the solid matrix or is present in an insoluble form. After thermal treatment, increasing in N might be due to the degradation of protein molecules [13]. Protein consists of a long chain of amino acid and it is tight coils. During the thermal treatment, as the protein in food waste was heated thus the tight coils will unwind. At this point, the protein was denatured, becomes smaller and less complex chains. [14] The correlation analysis illustrate that there was no statistical significant, strong positive relationship between temperature and percentage of N for 30 min (r = 0.571, p = 0.888) while for 60 min (r = 0.306, p = 0.549) there was no statistical significant, weak positive relationship between temperature and percentage. However, as illustrated in Figure 1 the percentage of N decreasing due, incomplete digestion during digestion step in TKN analysis. 3.1.2 Phosphorous. Normally in food waste, P can be found in protein food (milk, meat), nuts and seeds, vegetables and fruit and whole grain. The normal range of P in organic fertiliser was 0.04 % -8.65 % and it essential nutrient that been used for photosynthesis [12]. Higher concentration of P can be achieved with lower temperatures, but longer reaction times [5]. Figure 2 shows the graph with contact time 30 min the concentration of P is increasing to 366.30 mg/L and 52.93 mg/L as temperature increased to 60 o C and 70 o C. At 80 o C the concentration of P remains constant at 52.93 mg/l. However, as the temperature starts to increase to 90 o C and 100 o C the concentration of P slightly decreases to 52.46 mg/L and 51.70 mg/L correspondingly. While, graph with 60 min reaction time illustrate that as the temperature increase from 60 o C to 80 o C the concentration of P also increase until 49.96 mg/l but as temperature reach 90 o C and 100 o C the concentration drop to 31.86 mg/L. The concentration of P was increasing by 0.33 mg/L until 21.40 mg/L for all temperature and both reaction time compared to the untreated sample. Concentration of P was increasing due to the progressive disruption and disintegrate of chemical bonds in cell walls and membranes by thermal treatment [3]. Therefore, the intracellular organic material is released. At high temperature there will be a formation of insoluble phosphate. This clearly shows that thermal treatment with higher temperature will affect the concentration of phosphorous. This leads to the production of recalcitrant soluble organics hence reducing the biodegradability. The temperature of thermal treatment and percentage of P shows directly proportional with statistical significant for 30 min (r = 0.925, p = 0.000) and 60 min (r = 0.854, p = 0.042). This statistical indicate that increases in value of one variable, the second variable also increase in value. The highest concentration of phosphorous for 30 min and 60 min reaction time was at 80 o C. For 30 min it increases to 52.93 mg/L and at 60 min it increases to 49.96 mg/L compared to the untreated sample which only 31.53 mg/L. Even though the percentage of P in food waste was found to increase due to thermal treatment but the percentage is too small which can be considered there are no P in organic fertiliser. The highest percentage of phosphorous after thermal treatment is 0.0053 % where the normal range of phosphorous in organic fertiliser is 0.04% -8.65% [12]. Therefore, this food waste needs to undergo further treatment to increases the amount of P in organic fertiliser. 3.1.3 Potassium. The sources of K in the food sample were fruit and vegetables. 1.29% -6.94% is the range of percentage of potassium in organic fertiliser [12]. Figure 3 shows, the concentration of K in food waste for both reaction time were decreased drastically at 60 o C compare to untreated sample and it continuously decreases at 70 o C. However for the graph with 30 min reaction time, the concentration gradually increase from 80 o C until 100 o C.  Figure 3. Potassium for untreated and treated food waste.
While for graph with 60 min reaction time illustrate that concentration of K increase by 0.0276 mg/L during 80 o C but decreases slightly at 90 o C and 100 o C. K in food waste present in the form of potassium ions (K + ). During the thermal treatment, cells break down and release K + free and easily move into the water. K + are extremely soluble in water. Therefore the K + remained in the water since they were highly soluble in it [15]. The concentration of potassium at 60 o C for both reaction time were higher compared to other temperature. This is because there only a small amount of cell that been break down. Therefore more K in the solid sample and only little cells break down. For reaction time 30 min the correlation analysis shows no significant, strong positive of temperature and the concentration of K ( r = 0.980, p = 0.202). While for 60 min (r = 0.370, p = 0.183) there were no statistically significant, weak positive relationship. Thermal treatment using hot water bath is not suitable for potassium because this treatment could not achieve the minimum requirement of potassium for organic fertiliser. It shows decreasing in concentration of potassium compare to untreated sample and the percentage of potassium did not exist in an ideal ratio to provide nutrient for plant as organic fertiliser. Thermal treatment showed that potassium content failed to increase the level in the food waste.

Conclusions
From the research, we can conclude several conclusions which are: Percentage of N was in the range of organic fertiliser and can directly be employed. While for percentage of P its too small. Therefore it need to undergo further treatment to increase the percentage of P. However for K, hot water bath method is not suitable for potassium.
The statistical analysis two-ways ANOVA shows that there is an interaction effect between the independence variables (temperature and reaction time) and macronutrient (N,P) in food waste after thermal treatment. However, for potassium the interaction effect was not significant.


extfieldsofstudy: Biology Medicine

s2fieldsofstudy:
Agricultural And Food Sciences
Biology

text:
Identification and Validation of Candidate Genes Involved in Fatty Acid Content in Oil Palm by Genome-Wide Association Analysis

Oil palm (Elaeis guineensis) is the highest yielding oil crop per unit area worldwide, but its oil is considered unhealthy for human consumption due to its high palmitic acid content (C16:0). In order to facilitate breeding for fatty acid content in oil palm, genome-wide association analysis (GWAS) was used to identify and validate single-nucleotide polymorphism (SNP) markers and underlying candidate genes associated with fatty acid content in a diversity panel of 200 oil palm individuals. A total of 1,261,501 SNP markers previously developed using SLAF-seq (specific locus amplified fragment sequencing) were used for GWAS. Based on this analysis, 62 SNP markers were significantly associated with fatty acid composition, and 223 candidate genes were identified in the flanking regions of these SNPs. We found one gene (acyl-ACP thioesterase B genes) that was involved in fatty acid biosynthesis and that was associated with high palmitic acid content in the mesocarp. Over-expression of this gene caused a significant increase in palmitic acid content. Our study provides key loci that can be used for breeding oil palm cultivars with low palmitic acid content.

INTRODUCTION
Oil palm (Elaeis guineensis, 2n = 32) is an important tropical oil crop. Among all oil crops, oil palm has the highest oil yield per unit area: its oil yield is more than 6 times that of peanut, 8 times that of soybean, and 10 times that of rapeseed (Huang, 2017). The world production of palm oil in 2017 was approximately 65 million tonnes (http://faostat3.fao.org/home/E). The two oil storage tissues of oil palm are the mesocarp and kernel, each of which produces oil with a different fatty acid composition. Palm oil usually refers to the oil extracted from the oil palm mesocarp, where palmitic acid (16:0) is the major fatty acid (50%). By contrast, in kernel oil, lauric acid (12:0) is the major fatty acid (50%). A principal objective in oil palm breeding is to decrease the palmitic acid content (16:0) and to increase the oleic acid (18:1) and linoleic acid content (18:2) in the oil product. Recently, by using conventional breeding methods, researchers in Malaysia have developed an oil palm variety with high oleic acid (approximately 52%), although this oleic acid content is still far lower than that in peanut (>75% in high oleic acid varieties) or rapeseed (>80%) (Rajanaidu et al., 2017). Improving fatty acid composition via conventional breeding, however, is a slow process in oil palm because of the plant's long life cycle. The identification of genes controlling oil composition and the subsequent production of molecular markers linked to low palmitic acid content would be extremely helpful in accelerating the breeding and selection of oil palm.
In Arabidopsis, substantial research has been done to elucidate genes involved in the fatty acid biosynthesis pathway, which occurs mainly in the plastids and endoplasmic reticulum. Initially, acetyl CoA carboxylase (ACCase) catalyzes the conversion of the precursor converacetyl-CoA into malonyl-CoA (Sasaki et al., 1997). Catalyzed by ketoly-ACP synthase (KAS), malonyl-CoA is subsequently polymerized at a frequency of two carbons per cycle into the acyl carbon chain and is combined with acyl carrier protein (ACP) (Choi et al., 2000). Finally, the elongation of the carbon chain is terminated as catalyzed by acyl-CoA thioesterase (FAT) (Hunt et al., 2002). Previous research has shown that FAT exists as FATA and FATB (Salas and Ohlrogge, 2002). FATA was thought to be able to bring about the termination of C18:0-ACP and C18:1-ACP (Mekhedov et al., 2002). FATB, however, may also be involved in the termination of saturated fatty acyl-ACP (Bonaventure et al., 2004). In Arabidopsis thaliana, overexpression of the AtFatB1 gene can significantly increase palmitic acid content. The disruption of FatB expression resulted in a 56% reduction in palmitic acid and a 50% reduction of stearate in Arabidopsis seeds (Wilson et al., 2001;Buhr et al., 2002). Moreover, enhanced expression of FatB from Umbellularia californica was observed to significantly increase the lauric acid content (C12:0) in Brassica napus (Voelker et al., 1996) (Figure 1).
Association mapping is an efficient method for identifying molecular markers associated with agronomic traits based on natural populations with or without pedigree relationships (Flint-Garcia et al., 2003). The resolution of an association study depends on the extent and structure of linkage disequilibrium (LD) in the selected population. With the development of sequencing technology and high-throughput single-nucleotide polymorphism (SNP) markers, association mapping has been widely applied in different crops to dissect complex agronomic traits (Atwell et al., 2010;Huang et al., 2011;. However, there is still the problem of how to reduce the detection of spurious associations between traits and markers resulting from population structure. One solution is to perform association analysis between allelic and phenotype variation in a less structured population (Pritchard et al., 2000). Another solution for structured populations is to apply a mixed-model approach, which can decrease spurious associations (Sorkheh et al., 2008). Compared with QTL (quantitative trait loci) mapping in biparental cross populations, association analysis using natural populations is more time-and cost-effective, especially for woody perennial crops that have long life cycles and/or that require large planting areas. In oil palm, QTL mapping has been used to resolve the genetic bases of several complex traits, including yield components (Rance et al., 2001;Billotte et al., 2010;Jeennor and Volkaert, 2014;Tisne et al., 2015), fatty acid composition (Singh et al., 2009;Montoya et al., 2013), sex ratio (Ukoskit et al., 2014), and embryogenesis (Ting et al., 2013). Unfortunately, the contribution of QTL mapping studies to marker-assisted breeding outcomes has been less than expected. Although association mapping has been validated to be a reliable method for identifying trait-associated markers for marker-assisted selection (MAS), this method has rarely been applied to oil palm.
In this study, we used genome-wide association analysis (GWAS) analysis to identify SNP markers associated with fatty acid contents and identified candidate genes adjacent to these SNP markers based on our previous characterization of the genes involved in lipid metabolism pathways in oil palm (Xiao et al., 2019). We then analyzed the expression patterns of these candidate genes in different tissues. To validate the role of top candidate genes in regulating palmitic oil content, we transferred these genes into A. thaliana and over-expressed them. Our results provide a comprehensive understanding of fatty acid content in oil palm, and the SNPs and candidate genes detected will facilitate breeding for fatty acid content in oil palm.

Extraction and Measurement of Fatty Acid Contents in Oil Palm Mesocarp Tissues
Three fruits per oil palm individual (three biological replicates) were harvested, and fatty acid extraction and analyses for each mesocarp tissue were performed in triplicate (three different extractions as technical replicates). Approximately 60 mg of mesocarp (or 5 mg Arabidopsis seed) was used for extracting fatty acid according to methods described by Li-Beisson et al. (2013). Fatty acid composition was subsequently examined and measured using gas chromatography (Agilent DB-23, 30 m × 250). The heating procedure was as follows: the initial temperature was 180°C, followed by a temperature increase to 220°C at a rate of 10°C increase per minute. The gas pressure was 17.392 psi. The nine values (three biological replicates × three technical replicates) obtained per oil palm individual were averaged for subsequent association mapping. The contents of decanoic acid (C10:0), lauric acid (12:0), myristic acid (C14:0), tripalmitelaidin acid (16:1), palmitic acid (16:0), stearic acid (18:0), oleic acid (18:1), linoleic acid (18:2), and oil were determined with reference to an internal standard. The calculation formula was as follows: 1) total fatty acid = (total area of all measure fatty acid peaks × quantity of heptadecanoic acid-methyl ester)/(peak area of heptadecanoic acid-methyl ester × quantity of quantity of the sample); 2) relative fatty acid percentage = peak area of a specific fatty acid/total area of all measured fatty acid peaks. The peak area was calculated using the Agilent software.

SNP Genotyping
A total of 1,261,501 reliable SNPs markers (minor allele frequency > 0.05 and integrity > 0.8) were previously identified based on SLAF-seq (specific locus amplified fragment sequencing) by an Illumina HiseqTM 2500 in a diversity panel of 200 oil palm individuals . The SLAF tag sequences were mapped to the African oil palm genome (Singh et al., 2013, the assembled oil palm genome of Version 5) using a mapping software: the Burrows-Wheeler alignment tool (Li and Drubin, 2009).

Population Structure
Bayesian clustering was applied to analyze the population structure of 200 oil palm individuals using the software STRUCTURE (Pritchard et al., 2000). Based on the same set of SNPs, the number of subgroups (K) was predicted from 1 to 10, and the number of ancestors was determined according to the position of the minimum value, with an error rate obtained from 5-fold cross-validation. Maximum likelihood estimates for the ancestry proportion from each K subgroup for each accession were calculated. Five subgroups were indicated based on crossvalidation errors .

Association Mapping
To reduce spurious associations between alleles and phenotypic variation, mixed linear models (MLM) were used. Fixed effects were calculated using a Q (population) value matrix, and random effects were computed using a K (kinship) matrix. The Q+K value matrix was added to the MLM model. The Q matrix was obtained using STRUCTURE software (Pritchard et al., 2000), and the K matrix (genetic relationships among the 200 oil palm individuals) was obtained using SPAGeDi software (Hardy and Vekemans, 2002). Genome-wide association was performed using Tassel 5.0 (Bradbury et al., 2007). P-values for associations between SNP markers and fatty acid content were computed using the following formula: where Xβ represents the fixed effects other than the SNP under testing and the population structure; y is a vector of phenotypic observation; α is a vector of SNP effects; v is a vector of population effects; u is a vector of polygene backgroup effects; e is a vector of residual effects; Q is a matrix from backgroup effects relating y to v; and X, S, and Z are incidence matrices of 1s and 0s relating y to β, α, and u, respectively (Yu et al., 2006). Quantile-quantile plots (Q-Q plots) were drawn using the "ggplot2" software R package (Ginestet, 2011), and the Manhattan plots was drawn using the "qqman" software package (Turner et al., 2014). A total of 237,714 SNP markers, which had no missing data in the 200 oil palm individuals, were selected for association analysis. The threshold value (P < 4.2e −7 , −log 10 P approximately equal to 6.3) was set for detecting reliable trait-associated markers at a cutoff FDR (false discovery rate) of 0.1 (Benjamini and Hochberg, 1995). Meanwhile, when FDR was set as 0.01, the threshold value of P value and −log 10 P are 4.2e −8 and 7.3, respectively.

Prediction of Candidate Genes Associated With Fatty Acid Content
All genes in LD blocks (r 2 > 0.6) containing SNPs that were significantly associated with traits were identified for further candidate gene selection (Raman et al., 2015). The LD blocks present in the 200 oil palm individuals were estimated using the software Haploview v4.2. The number and size of the LD blocks on each chromosome were calculated according to previously established methods (Barrett et al., 2005). If SNP markers significantly associated with traits were located outside LD blocks, candidate genes were selected for further analysis following the criteria used by Zhou et al. (2017). Amino sequences of these selected genes were aligned using BLAST against the Arabidopsis protein database to predict the potential function of candidate genes. All candidate genes were selected based on gene ontology (GO) terms related to fatty acid biosynthesis and metabolism, and all transcription factors were selected based on Clusters of Orthologous Group of proteins (COG) within SNP-tagged genome regions.

Vector Construction and Transformation for Over-Expression of EgFatB
Among the candidate genes, acyl-ACP thioesterase B genes EgFatB1 and EgFatB2 were significantly associated with high palmitic acid contents in the mesocarp. Primers used for EgFatB gene cloning were designed using the Snapgene Viewer software (Table 1). Polymerase chain reaction (PCR) amplifications were performed in 50 µl reaction mixtures containing a 500 ng cDNA sample from the oil palm mesocarp, 1 × PCR buffer, 2 mM MgCl 2 , 5 U of TaqDNA polymerase (TaKaRa, China), 0.5 μM of each primer, and 0.2 mM dNTP mix. The PCR program included denaturation at 98°C for 30 s, followed by 30 cycles at 98°C for 10 s, 55°C for 30 s, and 72°C for 60 s, and a final extension at 72°C for 5 min. The PCR products were electrophoretically visualized on a 1% agarose gel and recombined into the pBinGlyRed3 vector (containing DsRed as a reporter gene and 35S as a promoter), which was then digested with EcoR I and XhoI. The constructed vectors were transformed into competent Escherichia coli cells (line Dh5l), and inserts were validated by sequencing. A plasmid with the correct sequence insert was transformed into Agrobacterium GV3101 and then transformed into A. thaliana using an Agrobacterium-mediated in planta transformation approach (Clough et al., 1998). The detailed protocols were as follows: the transformed Agrobacterium strain was streaked on solid LB medium containing Gent and Kana antibiotic overnight (28°C) and then a single validated colony was propagated in the liquid LB medium (28°C, 250 r/min) for 16 h. The turbid Agrobacterium liquid (300 ml) was then centrifuged at a rotation speed of 5000 r/min for 20 min, and the precipitation was suspended by a solution with 5% sucrose and 0.001% SilvetL-77 after removing the liquid LB medium. Arabidopsis inflorescences at the 3-4 day post-bolting stage were immersed in the Agrobacterium suspension and covered with plastic wrap for 16 h in the dark. After 1 week, these steps were repeated to increase transformation efficiency. Positive transgenic Arabidopsis seeds were confirmed by detection of red autofluorescence and by PCR validation of targeted genes. Control and transgenic Arabidopsis plants were grown with a 16 h light/8 h dark photoperiod at 25°C. Fatty acid composition of transformed Arabidopsis plants was examined with the same protocol as was used for oil palm mesocarp tissue.

Fatty Acid Composition in the Oil Palm Population
To perform an association analysis between SNP markers and relative fatty acid percentage, we first determined the relative fatty acid percentage of each of the 200 oil palm individuals from different geographical origins (Figure 2). Among the 200 oil palm individuals, relative palmitic acid percentage ranged from 31.3 to 48.8% with an average of 42.0%, oleic acid ranged from 31.3 to 50.1%, linoleic acid ranged from 7.1 to 18.5%,  and total oil content ranged from 29.8 to 70.3%. Relative palmitic acid percentage (C16:0) was negatively associated with stearic acid (r = −0.627; P < 0.001) and relative oleic acid content (r = −0.657; P < 0.001). Relative oleic acid percentage was also negatively correlated with linoleic acid content (r = −0.689; P < 0.001).

Genome-Wide Associations Between SNPs and Fatty Acid Content
To detect associations between allele variation and fatty acid content, we used MLM for GWAS; this controlled for the presence of population structure in the analyzed oil palm population. Meanwhile, five subgroups were previously indicated based on cross-validation errors ) (see Supplementary  Table 1 for Q values). The MLM analysis revealed 62 SNP markers that were significantly associated (P < 4.2e −7 ) with fatty acid content, including palmitic acid content (32 SNPs), oleic acid content (4 SNPs), linoleic acid content (1 SNP), and total oil content (25 SNPs) (Figures 3 and 4).

Identification of Candidate Genes Related to Fatty Acid Composition in E. guineensis
Of the 62 SNP markers significantly associated with fatty acid content, 6 were located in different LD blocks (r 2 > 0.6), while the other SNP markers were not located in defined LD blocks. A total of 223 candidate genes were identified in the flanking regions of SNP markers that were significantly associated with different fatty acid contents (Supplementary Table 2). Based on GO annotation results, one candidate gene was involved in fatty acid biosynthesis: an acyl-ACP thioesterase B gene (FatB, involved in the termination of the fatty acid chain).

Candidate Gene Expression in Different Oil Palm Tissues
The expression levels of candidate genes in fatty acid biosynthesis and metabolism pathways in different tissues and at different mesocarp developmental stages were estimated using RPKM values based on the method of Mortazavi et al. (2008). One candidate gene involved in fatty acid biosynthesis and metabolism was identified based on GO annotation results, and this gene showed higher expression levels in the mesocarp tissues than in other tissue types ( Figure 5A). The RPKM values for this gene were 41.8 and 37.9 at 10 and 15 weeks post-anthesis.
One FatB gene was detected as near to one significant SNP (EgChr7, 1731788). FatB genes have been reported to catalyze the termination of saturated fatty acyl-ACP. Interestingly, the expression level of EgFatB1 was generally higher in the mesocarp than in the kernel, especially at 10 and 15 weeks post-anthesis.

Candidate Gene Expression Profiles in Different Oil Palm Individuals
In order to analyze the expression levels of candidate genes in different oil palm individuals with different fatty acid contents, we performed RNA-Seq on eight oil palm individuals. Four oil palm individuals were chosen on the basis of higher palmitic acid content (45.8%, 43.6%, 43.9%, and 41.3%) and showed FatB gene RPKM values of 52.8, 133.6, 3.45, and 49.9, respectively ( Figure 5B). Four additional oil palm individuals were chosen on the basis of lower palmitic acid content (37.1%, 32.5%, 33.1%, and 30.3%) and showed FatB gene RPKM values of 37.1, 12.2, 29.9, and 11.6, respectively. The average expression level of FatB in oil palm individuals with higher palmitic acid content was two-fold higher than that of individuals with lower palmitic acid content.

Functional Validation of Egfatb1
The full length coding sequence of EgFatB1 was cloned and inserted into the pBinGlyRed3 vector. Genes inserted into this vector are promoted by a 35S promoter and in the same frame as the red autofluorescence reporter gene (DsRed3). The EgFatB1 over-expression plasmid was transformed into Arabidopsis. Positive transformants were screened in the T1 generation by detection of red autofluorescence in the seeds and by PCR validation. Positive transgenic plants in the T2 generation were investigated for fatty acid contents. Transgenic plants with overexpressed EgFatB1 increased the palmitic acid and stearic acid content but decreased the oleic acid and linoleic acid content in the seeds (Figure 6).

DISCUSSION
Oil palm (E. guineensis) is an important crop for human nutrition, with tens of millions of tonnes of palm oil consumed every year. Although breeding could potentially improve the nutritional value of palm oil for human consumption (e.g. by lowering the saturated fat content), breeding is hindered by the lack of genetic and genomic resources available for this crop. Using SLAF-seq technology, we genotyped a diversity panel of 200 oil palm individuals from four countries to obtain 1.2 million genome-wide SNPs . We associated these SNPs with fatty acid content and identified dozens of candidate genes involved in fatty acid biosynthesis and metabolism pathways. One gene, EgFatB1, which was highly and specifically expressed in the mesocarp, was validated using Arabidopsis transformation: over-expression of EgFatB1 resulted in increased saturated fat content in Arabidopsis seeds.
Oil palm (E. guineensis) originated from West and Southwest Africa, especially in the area between Angola and Gambia. Oil palm is well-known for its highest oil yield per unit area. Palm oil is an edible vegetable oil and derived from the mesocarp (reddish pulp) of the oil palm fruit. Along with coconut oil, palm oil is semi-solid at room temperature due to a high content of saturated fatty acid, especially palmitic acid (C16:0), which accounts for approximately 50% of palm oil fatty acid content. However, how fatty acid composition varies in different oil palm germplasm is unknown. In this study, we investigated the variation of fatty acid composition among 200 oil palm individuals. A large variation in fatty acid composition was detected between these oil palm individuals. Our study identified useful oil palm germplasm for improving fatty acid composition, especially for decreasing palmitic acid content (lowest value: 31.3%) and increasing oleic acid content (highest value: 50.1%).
GWAS was firstly used as a powerful method to identify a broad range of complex diseases in human populations (Burton et al., 2007). Subsequently, with the completion and availability of additional genome sequences, this approach was used in flowering plants, including rice (Huang et al., 2011), foxtail millet (Jia et al., 2013), maize , and Arabidopsis (Atwell et al., 2010). There are, however, some limiting factors in association analysis, such as insufficient marker density and population structure effects (Cappa et al., 2013). In a previous study, SLAF technology was used to develop a large number of SNP markers and to evaluate potential population structure, thus addressing these limiting factors and providing useful markers for MAS in oil palm. Palm oil accounts for 25% of vegetable oil traded worldwide annually and uses 5% of the global oil planting area. However, breeding progress in this tropical species is still slow, mostly due to the long life cycles of individual plants. Currently, only five to six generations of phenotype-based selection have been completed since the 1920s and 1930s (Hartley, 1967).  MAS has major potential to speed up the breeding of oil palm cultivars and reduce the length of conventional breeding cycles. Identifying trait-associated markers is a prerequisite for MAS. In the last decades, molecular markers have been used to identify QTL associated with important agronomic traits. Most marker development is based on conventional methods that result in modest density, including restriction fragment length polymorphism (RFLP) (Mayes et al., 1997), amplified fragments length polymorphism (AFLPs) (Singh et al., 2009), and simple sequence repeat (SSR) marker types (Billotte et al., 2010). To increase marker density, SNP markers were developed in oil palm. These high density SNP markers were successfully used in our study to identify molecular markers associated with important agronomic traits.
In oil palm, the first genetic linage map was constructed using RFLP markers (Rance et al., 2001). QTL mapping was subsequently used to decipher the genetic bases of yield components (Rance et al., 2001;Billotte et al., 2010), fatty acid composition (Singh et al., 2009;Montoya et al., 2013), and sex ratio (Ukoskit et al., 2014), among other traits. These QTL mapping experiments involved populations produced from one to four parents. Montoya et al. (2013) reported 19 QTLs associated with fatty acid composition in palm oil using a backcross population of Elaeis oleifera with E. guineensis. Acyl-ACP thioesterase A and Stearoyl-ACP desaturase genes were found located in these QTL intervals. However, the confidence interval of these QTLs ranged from 6 to 30 cM, or approximately 6-30 Mbp of physical genome sequence in oil palm (Montoya et al., 2013). Teh et al. (2016) identified three key loci for high mesocarp oil content in oil palm, which were located on Chr5, Chr9, and Chr11. In our study, we identified 62 SNP markers (P < 4.2e −7 ) significantly associated with fatty acid content on Chr3, Chr5, and Chr11. Meanwhile, we also identified SNP markers associated with fatty acid on Chr1, Chr6, Chr10, Chr12, and Chr13. The EgFatB1 gene was identified on Chr6 and validated to be associated with variation in palmitic acid content.
In our study, some SNP markers were associated with variation in fatty acid content, which is useful for selecting low palmitic acid genotypes in oil palm breeding and subsequently improving the nutritional value of palm oil. Moreover, there have been some previous examples where fatty acid composition in different crops has been improved using various biotechnology and mutation approaches. In rapeseed, the relative oleic acid content was increased to 71% by EMS and 60 Co mutagenesis (Rucker and Robbelen, 1995). In soybean, oleic acid content increased to 85% in a fad2-1 mutant (Wagner et al., 2011). Meanwhile, in peanut, Norden et al. (1987) bred a high oleic acid cultivar (F435) from a natural mutant (Norden et al., 1987). Hence, artificial mutagenesis in oil palm could produce germplasm with significant divergence in fatty acid composition. In the present study, FatB was validated as a major effect gene for relative palmitic acid content, allowing us to screen for low palmitic acid content in oil palm by selecting for FatB mutations. The availability of molecular markers associated with agronomic traits will allow breeders to rapidly identify target traits in seedlings, thus accelerating selection and breeding schemes. Meanwhile, molecular markers associated with agronomic traits can also be used to detect the introgression of the trait of interest into elite varieties from more diverse germplasm.

DATA AVAILABILITY STATEMENT
The raw SLAF-seq data for the 200 oil palm individuals has been deposited into the European Nucleotide Archive (http://www. ebi.ac.uk/ena). The bioproject number is PRJEB26466.

AUTHOR CONTRIBUTIONS
YX, WX, and CZ participated in the design of the study. YX and WX performed the statistical analysis and drafted the manuscript. JW and YD were involved in analysis of the added transcriptome datas. AM critically revised the manuscript. TL and WZ did the major experimental work including the extraction and measurement of oil content and relative fatty acid contents. WT, YD, DH, and XH contributed to and advised on the statistical analysis. All authors read and approved the final manuscript.