MINISTRY OF EDUCATION AND TRAINING CAN THO UNIVERSITY SUMMARY OF DOCTORAL DISSERTATION Major in Soil Science Identification code: 9620103 NGUYEN THI PHUONG COMPOSTING MICROBIAL ORGANIC FERTILIZER OF SLUDGES FROM WASTEWATER TREATMENT PLANTS OF BEER AND SEAFOOD PROCESSING FACTORIES Can Tho, 2019 This study was achieved at Can Tho University Scientific Supervisor: Assoc Prof Dr Nguyen My Hoa This dissertation was defended at the University Examination Council At: …………………………………… At…hour , on date…month….year… Reviewer 1:…………………………… Reviewer 2:…………………………… Reviewer 3:…………………………… The dissertation is available at: Learning Resource Center, Can Tho University National Library of Viet Nam PUBLICATIONS RELATED WITH THE DISSERTATION [1] Nguyen Thi Phuong, Nguyen My Hoa, Do Thi Xuan, Lam Ngoc Tuyet, Vo Thi Thu Tran, 2016 “Characteristics of sludges from wastewater treatment plants of beer and seafood processing factories” Journal of Science, Can Tho University, 45A/2016, p.74-81 (In Vietnamase) [2] Nguyen Thi Phuong, Nguyen My Hoa, Do Thi Xuan, Lam Ngoc Tuyet, 2017.“Composting of sludge from wastewater treatment plants of seafood processing factories” Science and Technology Journal of Agriculture & Rural Development, 5, p.54-61 (In Vietnamase) [3] Nguyen Thi Phuong, Nguyen My Hoa, Do Thi Xuan, Lam Ngoc Tuyet, 2017 “Composting of sludge from wastewater treatment plants of beer processing factories” Journal of Vietnam soil science, 50, p.47-52 (In Vietnamase) [4] Nguyen Thi Phuong, Nguyen My Hoa, Do Thi Xuan, 2018 " Effect of micro - organic composts from beer and seafood sludge on growth and yield of Okra (Abelmoschus esculentus)" Journal of Agricultural Science and Technology of Vietnam, 2(87), p.7-10 (In Vietnamase) [5] Nguyen Thi Phuong, Nguyen My Hoa, Do Thi Xuan, 2018 “Production and assessment efficiency of microbial-organic fertilizers from beer and seafood factories’ sludge on vegetable yield” Journal of Science, Can Tho University, special issue Agriculture 54 p.81-90 (In Vietnamase) [6] Nguyen Thi Phuong, Nguyen My Hoa, Do Thi Xuan, 2018 “Effect of microbial organic fertilizers from seafood sludge on growth and yield of winter melon (Benincasa hispida)” Journal of Vietnam soil science, 54, p.19-24 (In Vietnamase) CHAPTER INTRODUCTION Rationale of the study In many wastewater treatment facilities, the bottleneck of the sludge handling system is the dewatering operation Beer (BS) and seafood sludges (SS) were effluents dewatered in waste water treatment plants of beer and seafood factoires The annual sludge production from beer breweries and seafood factories is about million tons and 313,170 tons, respectively (Fillaudeau et al (2006); Ministry of Industry and Trade, 2009, 2016; Vo Phu Duc, 2013 ) According to Senthilraja et al (2013), Feng et al (2008), and Vo Phu Duc (2013) beer and seafood sludges are nontoxic and contained much of the organic matter and valuable nutrients For these reasons, recycling of sludges for agricultural purposes seems to be an appealing solution that enables using of valuable components and resolve pollutants ( Ministry of Natural Resources and Environment, 2013; Oanh and Dieu,2016; Saviozzi et al., 1994; Stocks et al., 2002; Thomas and Rahman, 2006) Both BS and SS possessed poor compressibility and settleability (Jin et al., 2003; Li et al., 2012) Therefore, it is necessary to mix BS and SS with organic materials such as sugarcane sludge, bagasse and straw that are high in cellulose and porosity to provide energy for microorganisms during composting process, and increase compost quality However, the most suitable ratio mixture of sludge and organic materials to produce high quality microbial-organic fertilized to meet the standard of Vietnam and to increase crop yield is still questionableAt present, there are not many research papers on beer and seafood sludges for production of microbial-organic compost (Vo Phu Duc, 2013; Oanh and Dieu, 2016) In addition, isolating fungal strains from rice straw, bagasse and coconut fiber was also carried out to find out new fungi that can decompose organic materials, resisted to pathogenic fungi and promote organic composting process Because of these issues, study on production of microbial-organic fertilizers from beer and seafood factories’ sludge is necessary as an effective treating of sludge 1.2 Objectives of study Determination of optimal composting formula for the production of microorganic compost from beer and seafood sludges; 1.1 Assessment effetiveness of microbial-organic fertilizers from beer and seafood factories’ sludge on vegetable yield; Isolation and selection of fungal strains capable of decomposing organic materials for further use as a microbe in production microbial organic fertilizer 1.3 Research activity Determination of physical, chemical, nutritional and biological characteristics of BS and SS Efficiency of dried beer and seafood sludge on growth and yield of vegetable grown under the greenhouse condition; Determination of decomposability and suitable composting ratio of beer/seafood sludge and organic materials at bag scale; Production microbial-organic fertilizers from beer and seafood sludge at scale of 0,5 meter cubic; Assessing efficiency of microbial-organic fertilizers from beer and seafood factories’ sludge on vegetable yield, consisting of mustard, okra, cucumber, and winter melon in the field conditions; Isolation and selection of organic material decomposable-fungal strains 1.4 Scope of the study The study focused on sludges from wastewater treatment of beer production and seafood processing plants in the Mekong delta to produce microbial-organic fertilizer using Trichoderma spp from Trico-DHCT product as the beneficial microorganism The crops used for testing the effect of microbial-organic fertilizer were vegetables for short growth duration The isolation of the fungal strain focused on organic material decomposability of the fugus for the aim of the wide use in production of microbial-organic fertilizer 1.5 Scientific significance and applicability of study The results showed that BS and SS are two sources rich in nitrogen, phosphorus, micro nutrients and not contain heavy metal contaminants Sugarcane cake was suggested to mix with beer/seafood sludges for the production of bio-organic fertilizer The optimal mixing ratio of sludge and sugarcane sludge with 20:80 is recommended for the production of microbial-organic fertilizers with high quality The research has proved the efficiency of microbial organic fertilizer produced from both sludges on vegetable yields such as mustard, okra, cucumber, and winter It is recommended to use tons /ha of microbial organic fertilizer from the sludges with recommended dose of inorganic fertilizer to increase vegetable yield Four strains of fungi which possessed organic material decomposability and fungal pathogens antagonistic character were selected Two strains were identified as Neurospora crassa and Neurospora intermedia, which decomposed well mixture of sludge and sugarcane cake with ratio of 20:80 .CHAPTER METHODOLOGY 2.1 Determination physical, chemical, nutrient and biological characteristics of beer and seafood sludges Sludge samples were collected at beer factories in Soc Trang, Tien Giang, and Bac Lieu provinces and at seafood processing factories in Dong Thap, An Giang, Hau Giang, Tien Giang, and Bac Lieu provinces Sugarcane cake and bagasse were collected at Hau Giang sugarcane factory Cow dung was collected from Long Hoa dairy cooperative Analytical methods of input materials are presented in below detail (Table 2.1) Table 2.1 Analytical methods of input materials Analytical methods Parameter Unit Moisture % Dry at 105oC to constant weight Samples are taken in a 100 cm3 box Bulk density g/cm3 and then dried at 1050C for 24 hours Extract with water with a soil ratio of pHH2O 1: Measure by pH meter Extract with water with a soil ratio of EC mS/cm 1: Measure by EC meter The samples were dried at 1050C for Organic cabon %C hours and at 8300C for hours Anaerobic digestion with salicylic acid + Total nitrogen %N concentrated H2SO4 + H2O2 and determined by Kjeldahl method Anaerobic digestion with salicylic acid + concentrated H2SO4 + H2O2 and Total phosphorus %P2O5 colorimetric on spectrophotometer at 880nm Total potassium %K2O Anaerobic digestion with salicylic acid + N available %N P available %P2O5 K available mg/kg Ca, Mg, Zn,Cu, Mn mg/kg concentrated H2SO4 + H2O2 and measured on atomic absorption Extract with H2SO4 0.5N (Vietnamese Standard 9295:2012) Extract with acid citric 2% (Vietnamese Standard 8559:2010) Extract with HCl 0.05N (Vietnamese Standard 8560:2010) Anaerobic digestion with concentrated H2SO4 + H2O2 measured on atomic absorption Vietnamese Standard 9291:2012 Vietnamese Standard 9290:2012 Vietnamese Standard 8467-2010 Vietnamese Standard 8882-2011 Cd mg/kg Pb mg/kg As mg/kg Hg mg/kg Ecoli, Coliforms, CFU/g Colony forming unit method Salmonella Dw 2.2 Efficiency of sun-dried beer and seafood sludges on growth and yeild of vegetable under the greenhouse condition 2.2.1 Germination of mustard (Brassica juncea) on substrates of sun-dried sludges Each sludge were put in a plastic tray with a thickness of cm Then each tray would directy exposure the Sun from am up to pm, and rotate the sample every 30 minutes The sludge wastes were treated by directly exposing under the sunshine at hours, hours and hours to get three levels of moistures i.e 10%, 30% and 50%, respectively to treat human pathogens The experiment was performed in trays with a completely randomized block with eight treatments (Table 2.2) and three replications for each The volume of soil, sun-dried sludges, organic fertilizer of sugarcane cake (SF) used for each tray is 0.5 kg/tray 100 green mustard were sowing into the trays and sprinkle with 60% humidity to ensure germination in all trays After 14 days, germinated percentage, height, fresh weight, dry weight of mustard were recorded Table 2.2: Substrates of sun-dried sludges on mustard germination Treatment Types of fertilizer Control (Soil) Beer sludge at 10% humidity (BS-10) Beer sludge at 30% humidity (BS-30) Beer sludge at 50% humidity (BS-50) Seafood sludge at 10% humidity (SS-10) Seafood sludge at 30% humidity (SS-30) Seafood sludge at 50% humidity (SS-50) Organic fertilizer of sugarcane cake (SF) 2.2.2 Evaluation efficiency of sun-dried sludges on growth and yield of green mustard (Brassica juncea) As a result of experiment at section 2.2.1, two samples of BS-30 and SS-50 were used as compost on growth and yield of green mustard The experiment was completely randomized in a pot with six treatments and three replications (Table 2.3) Each pot contains 7kg dry land The BS-30 and SS-50 mixed with sugarcane cake (SC), organic fertilizer of sugarcane cake used for each pot is tons/ha and distributed a week before sowing Green mustard seed is sown 10 seeds into pots and watered to maintain moisture Each pot would choose well three plants when the plant highthened about cm Fertilizers are used according to the recommended rate of Tran Thi Ba (1999) with 55N-32P2O5-46K2O (kg/ha) Table 2.3: Experiment on growth and yield of green mustard Mixture Ratio Order Treatment (%Dry weight) Control (soil) Soil+ (BS-30: SC) 20:80 Soil+ (BS-30: SC) 50:50 Soil+ (SS-50: SC) 20:80 Soil+ (SS-50: SC) 50:50 Soil+ SF Observed parameters after 30 days included plant height, leaves number/plant, fresh and dry weight, pathogens i.e E coli, Coliforms and Salmonella The analyzing method was described in section 2.1 2.3 Determination of decomposition ability, suitable composting ratio of sludges in the bag 2.3.1 Evaluation decomposable capacity of organic materials Beer sludge (BS) was collected from Tien Giang beer factory and seafood sludge (SS) was collected from Hau Giang seafood company Both sugarcan cake (SC) and bagasse were sampled from Vi Thanh sugarcane factory Experimental equipment and chemicals used department of soil science, Can Tho University, including of Trichoderma-DHCT fungus.The experiment was arranged in a completely randomized block of 13 treatments with replications for each treatment (Table 2.4) For incubation, g a mixture of sludge and organic materials at moisture of 65%, and placed in 150-mL plastic jars One vial containing NaOH solution (3N) were placed into 150-mL plastic jars After this, the vial containing the NaOH solution was renewed weekly Moisture was checked by weighing at each sampling date The amount of Trichoderma fungus was injected into each treatment with 100g /m3 (Dw) at initial compost After this, all of the jars were placed in the dark at a constant temperature of 300C for 45 days Carbon mineralisation was measured at 7, 14, 21, 30 and 45 day after incubation The CO2 was titrated with H2SO4 (0.01 N) Mass loss was determined by weighing the sample after 45 days of incubation Table 2.4: Treatments, mixture ratio and C/N ratio Order 10 11 12 13 Treatments Rice straw Bagasse Sugarcane cake Beer sludge Seafood sludge BS: SC SS: SC BS: SC:straw SS: SC:straw BS: SC:bagasse SS: SC:bagasse BS: SC:bagasse SS: SC:bagasse Ratio (%Dry weight) C/N 71 271 19 12 20:80 20 20:80 20 20:60:20 24 20:60:20 23 10:60:30 30 10:60:30 30 20:60:20 30 20:60:20 30 2.3.2 Composting beer and seafood sludges in bag condition The samples of BS, SS, rice straw, sugarcane cake, bagassem and cow dung were collected and disposed as similarly as sections 2.1.1 and 2.3.1 Incubation equipments consisted of plastic bags of 50kg, weighing scale of 30kg, canvas cover plastic bag, thermometer, and Trichoderma-DHCT fungus The experiment was arranged in a completely randomized block of 14 treatments with replications for each treatment (Table 2.5) The treatments were mixed by dry weight The amount of Trichoderma fungus was injected into each treatment with 100g /m3 (Dw) at initial and after 15-day incubation with 50 for each injection The basis on the results of section 2.3.1 and initial C/N ratio, combination sludge with sugarcane cake and straw or bagasse with ratio of 10:60:30; 10:70:20, 20:60:20, and 20:80 was studied Table 2.5 treatment and mixture ratio Order Treatments Ratio (%Dw) C/N BS: SC:bagasse 10:60:30 30 BS: SC:bagasse 10:70:20 23 SS: SC:bagasse 10:60:30 30 SS: SC:bagasse 10:70:20 23 Cow dung: SC:bagasse 10:60:30 21 Cow dung: SC:bagasse 10:70:20 20 BS: SC: rice straw 20:60:20 24 BS: SC: rice straw 10:70:20 21 SS: SC: rice straw 20:60:20 23 10 SS: SC: rice straw 10:70:20 20 11 Cow dung: SC: rice straw 20:60:20 23 12 Cow dung: SC: rice straw 10:70:20 21 13 BS: SC 20:80 20 14 SS: SC 20:80 20 Note: BS: Beer sludge, SS: seafood sludge, SC: sugarcane cake Dw: dry weight Parameters included temperature, humidity and weight loss were monitored weekly until 75 days after incubation At days 0, 49, 63, and 75 from initiation, one composite sample (~1 kg dry matter) of three subsamples was collected from each pile These samples were used for analyses of total C, N, pH, and EC These samples were also used for analyses of total N, phosphorus (P), potassium (K), amount of authors such as Jones et al (2011), Bui Thi Nga et al (2014), Lakhdar et al (2010), and Duong Minh Vien et al (2011) evidenced EC value did not affect plant and soil In addition, contains of total nitrogen and phosphorus in the both beer and seafood sludges were high, higher than other sludge sources such as sugarane sludge, pond sludge,sewage sludge but the level of potassium from the two these sludges reached the level of poverty The total nitrogen content (NT) in the sludge was high because malt, adjuncts, and nitric acid used for cleaning may contribute to the total nitrogen content (Olajire, 2012) Phosphorus can also come from cleaning agents The actual phosphorus concentration will depend on the water ratio and the cleaning agent used (Olajire, 2012) (Table 3.2) Table 3.2 Nutrients characteristics of material before conpost TN TP TK Na Pa Ka (%) (%P2O5) (%) (%) (%) (%) Rice straw 0.6 0.31±0.00 1.37 Bagasse 0.21 0.13±0.01 0.20 0.02 0.002 0.16 Sugarcane cake 2.31 6.37±0.01 0.78 0.45 3.37 0.48 Cow dung 1.31 3.76±0.00 1.18 0.22 2.87 1.08 BS-Soc Trang 3.95 4.99±0.02 0.2 0.29 1.93 0.16 BS-Tien Giang 2.61 10.7±0.03 0.97 0.18 2.38 0.10 BS-Bac Lieu 2.59 5.56±0.10 0.23 0.33 1.04 0.16 SS-Tien Giang 2.11 7.27±0.02 0.16 0.25 5.54 0.09 SS-Dong Thap 3.87 7.29±0.04 0.50 0.74 4.72 0.36 SS-An Giang 2.94 6.32±0.07 0.16 0.22 3.85 0.05 SS-Hau Giang 5.62 7.17±0.16 0.74 0.32 4.97 0.18 SS-Bac Lieu 4.65 4.66±0.02 0.45 0.27 3.53 0.3 Note: Mean ±SD, BS: beer sludge, SS: seafood sludge, TN: total nitrogen, TP: total phosphorus, TK: total potassium, Na: available nitrogen, Pa: available phosphorus, Ka: available potassium “-“: not data Materials The organic carbon content (% C) from both sludges was low, leading to low C/N ratio therefore, beer and seafood sludges should be mixed with organic materials with high fiber content such as sugarcane cake, bagasse and rice straw to increase porosity and supply carbon source for microorganisms growth during composting (Table 3.3) Table 3.3 Organic carbon and C/N ratio of materials Materials C (%) C/N Rice straw 42.67 71 Bagasse 57.94 276 12 Sugarcane cake Cow dung BS-Soc Trang BS-Tien Giang BS-Bac Lieu SS-Tien Giang SS-Dong Thap SS-An Giang SS-Hau Giang SS-Bac Lieu 31.78 46.66 21.53 31.75 31.38 42.09 41.71 34.24 42.81 37.43 14 36 12 12 20 11 12 8 Note: BS: beer sludge, SS: seafood sludge 3.1.2 Contains calcium, magnesium, micronutrients, heavy metals, and pathogenic microbes The results showed that contains of Ca, Mg, Mn, Zn, and Cu in both BS and SS were high and equal or higher than sugarcane sludge Furthermore, the nutrient content of micro elements got under standard limit of Vietnam 50/2013 / MoRE Therefore, these sludges provided additional useful sources for microbial organic composting (Table 3.4) Table 3.4 Calcium, magnesium, manganese, copper, zinc in materials Ca Mg Mn Zn Cu Materials (% CaO) (% MgO) (mg/kg) (mg/kg) (mg/kg) Bagasse 0.05 0.09 70 11 Sugarcane cake 4.44 0.61 327 256 106 Cow dung 2.35 1.31 664 567 159 BS-Soc Trang 0.84 0.72 359 132 454 BS-Tien Giang 1.23 0.42 436 1327 201 BS-Bac Lieu 1.13 0.84 293 144 514 SS-Tien Giang 4.78 0.01 114 104 13 SS-Dong Thap 3.72 0.13 174 272 53 SS-An Giang 5.41 0.15 154 771 74 SS-Hau Giang 3.02 0.39 293 349 340 SS-Bac Lieu 5.79 1.57 187 526 539 Note: BS: beer sludge, SS: seafood sludge The samples of BS and SS did not contain heavy metal toxic but the microbial population is E coli and Coliforms exceed the limit allowed under Decree 108/2017/goverment Decree for E coli and 13 Vietnam standard No 40/2011/MoRE for Coliforms Hence, microbial organic composting was essential to kill pathogens (Table 3.5 and 3.6) Table 3.5 Contains heavy metals in materials Cd Pb As Hg Materials (mg/kg) (mg/kg) (mg/kg) (mg/kg) Bagasse 0.01 1.58 Sugarcane cake 0.48 1.10 0.04 ND Cow dung 1.06 0.66 BS-Soc Trang 0.11 BS-Tien Giang 0.55 0.55 0.67 ND BS-Bac Lieu 0.15 SS-Tien Giang 0.08 0.37 SS-Dong Thap 0.12 0.45 SS-An Giang 0.38 1.10 SS-Hau Giang 1.18 0.09 0.09 ND SS-Bac Lieu 5.03 8.66 Threshold limit