Nghiên cứu quá trình tiền xử lý và hệ vi sinh vật phân giải vỏ quả cà phê vối (coffea robusta) để lên men tạo ethanol tt tiếng anh

MINISTRY OF EDUCATION AND TRAINING CAN THO UNIVERSITY SUMMARY OF DOCTORAL DISSERTATION Major: Food Technology Code: 62.54.01.01 LE P DO VIET PHUONGHAM TAN QUOC STUDY ON THE PRETREATMENT PROCESS AND THE MICROBIOTA FOR COFFEE PULP (Coffea robusta) HYDROLYSIS TO PRODUCE ETHANOL Can Tho, 2020 THE DISSERTATION WAS COMPLETED AT CAN THO UNIVERSITY Advisor: Assoc Prof Dr Le Nguyen Doan Duy Sub-advisor: Dr Pham Van Tanc Prof Dr Nguyen Van Muoi The doctoral dissertation was evaluated by the Committees at the basic level Meeting at: Meeting room 2, 2nd floor, Campus II – Control Hall, Can Tho University At: 14 p.m., date 17/11/2018 Reviewer 1: Assoc Prof Dr Dai Thi Xuan Trang Reviewer 2: Prof Dr Dong Thi Anh Dao You can find a copy of this dissertation at the library: Learning Resource Center, Can Tho University National Library of Vietnam LIST OF PUBLISHED WORKS Do Viet Phuong, Pham Van Tan and Le Nguyen Doan Duy 2017 Determination of caffeine in coffee pulp (Coffea robusta) using UVVisible spectrophotometer Vietnam Journal of Chemistry, 55: 86-91 (ISSN 0866-7144) Do Viet Phuong, Pham Van Tan and Le Nguyen Doan Duy 2017 Optimizing decaffeination conditions from coffee pulp in Vietnam (Coffea robusta) using hot water extraction Proceedings of the 15th ASEAN Conference on Food Science and Technology, 112-118 (ISBN 978-604-67-1007-3) Do Viet Phuong, Le Huong Thuy, Dam Sao Mai, Dang Thi Sau, Le Nguyen Doan Duy and Pham Van Tan 2018 Enzyme cellulase recovery from Trichoderma asperellum QT5 fertilized from coffee cultivation area in KrongBuk district, DakLak province, Vietnam Science and Technology Journal of Agriculture and Rural Development, 3+4: 150-159 (ISSN 1859-4581) Do Viet Phuong, Le Nguyen Doan Duy and Pham Van Tan 2019 Investigation of pretreatment, hydrolysing and fermentation processing from coffee pulp to form ethanol Science and Technology Journal of Agriculture and Rural Development, 5: 115-123 (ISSN 1895-4581) Do Viet Phuong, Le Pham Tan Quoc, Pham Van Tan and Le Nguyen Doan Duy 2019 Production of bioethanol from Robusta coffee pulp (Coffea robusta L.) in Vietnam Foods and Raw materials, 7(1): 10-17 (ISSN 2310-9599) CHAPTER INTRODUCTION 1.1 The rationale of the study In recent decades, the increasing demand for energy and the problem of environmental pollution caused by the use of fossil fuels has prompted countries to pay more attention to pursuing and replacing them with renewable energy sources and especially bioethanol production One of the most abundant sources of materials for bioethanol production is lignocellulosic biomass According to scientists' estimation, ethanol production from lignocellulose biomass could increase 16 times of the current yield (Kwon et al., 2013) Recent research indicates the great potential of coffee hulls waste in bioethanol production (Saha and Cotta, 2008) Burning ethanol instead of gasoline will reduce carbon emissions by more than 80% (Mussatto et al., 2011) However, the biggest barrier to production of ethanol from this source is the high hemicellulose and lignin content in it These substances are closely associated to cellulose and provide a barrier to protect the attack of chemicals as well as enzymes to the cellulose molecule (Ragauskas et al., 2006; Hahn-Hägerdal et al., 2006) Facing that difficulty, scientists have researched to find many methods of pretreatment of lignocellulose materials before conducting saccharification and fermentation Besides, lignin is known as one of the main constituents of plant cell walls and algae Lignin occurs most often in all types of woody stems, so it is also known as "wood" (Martone et al., 2009) In coffee pulp, lignin accounts for about 20.07% and when compared with lignin content in coffee pulp with some sources of lignocellulose biomass similar to: Lignin in straw accounts for 4.65%; lignin in corn cob accounts for 15%; in bagasse 20% or in grass 12 ÷ 18% (Sun and Cheng, 2002; Mosier et al., 2005; Goyal et al., 2008; Sassner et al., 2008; Zhang et al., 2009) It can be seen that the lignin content in coffee pulp is higher than some similar lignocellulose biomass sources and this will be a big obstacle to the pretreatment of coffee pulp before hydrolysis and fermentation ethanol Therefore, it is necessary to conduct surveys on pretreatment methods to remove this lignin and at the same time retain the cellulose content of the material before conducting the hydrolysis and fermentation process to create bioethanol In fact, the coffee production in Vietnam is concentrated mainly in the Central Highlands provinces so it will reduce the cost of collecting raw materials In addition, it is possible to realize the potential for huge reserves of coffee pulp in Vietnam, along with topical issues that are the problem of environment pollution, to find and replace fossil energy sources gradually exhausted Therefore, Robusta coffee pulp were selected as the raw materials for the study: "Study on the pretreatment process and the microbiota for coffee pulp (Coffea robusta) hydrolysis to produce ethanol" 1.2 Objective of the study Using acid, alkali, microwave or white rot fungi to pretreatment the coffee pulp to remove hemicellulose and lignin the most Concurrently, the cellulase enzyme was recovery from mold and applied to hydrolysis process The hydrolysate was fermented to compared the hydrolysis efficiency between the obtained enzyme and the commercial enzyme 1.3 Contents Investigate decaffeine, depolyphenol and pretreatment process of coffee pulp at the same time optimizing parameters for decaffeine and depolyphenol Recovery of crude cellulase enzyme from mold and preliminary purification Compare the hydrolysis efficiency between the recovery enzyme and the commercial enzyme At the same time, investigating the factors affecting the hydrolysis process and optimizing hydrolysis conditions to get the highest reducing sugar content Check the composition of hydrolysate and give a method to detoxify the hydrolysate Investigate factors affecting fermentation and methods of fermenting hydrolysate to create ethanol 1.4 Scientific and practical significance Researching and offering useful solutions to solve by-products problem in processing industry are research trends that are being paid attention at home and abroad Using Robusta coffee pulp is a practical option when Vietnam is the 2nd largest exporter of green coffee in the world (in particular, the world's largest Robusta coffee exporter) Research results from the thesis provided data on the chemical composition of the Robusta coffee pulp, showing the feasibility of using this by-product source as a potential lignocellulose biomass source for bioethanol production Research has focused on the use of solutions to limit the use of toxic high concentrations of chemicals by combining pretreatment methods 1.5 New contributions Propose a microwave-assisted pretreatment solution to significantly reduce the amount of using chemicals (only use dilute alkaline, dilute acid) that help reduce environment pollution and increase efficiency; the need to adjust the order of pretreatment steps according to the material composition Evaluate the formation and impact of a number of poisonous chemicals for fermentation and propose a solution to eliminate Recovery cellulase enzyme from Trichoderma asperellum (QT5) isolated from the surface of broken coffee pods, effectively applied in the hydrolysis of Robusta coffee pulp The information from these results was a scientific document for the study of bioethanol from sources of high lignin sub-products and used in teaching about management and utilization of sub-products in food production 1.6 Outline of the dissertation The dissertation consists of 142 pages with five chapters: Chapter 1: Introduction (pages: 1÷4); Chapter 2: Literature review (pages: 5÷51); Chapter 3: Materials and methods (pages: 52÷79 with 28 experiments); Chapter 4: Results and discussion (pages: 78÷141) and Chapter 5: Conclusions and suggestions (page 142) The primary content has 33 tables and 65 figures The dissertation consists of 262 references (236 English references and 26 Vietnamese references) CHAPTER LITERATURE REVIEW 2.1 Coffee pulp Coffee pulp are the first by-product obtained during the processing of wet coffee beans The coffee pulp account for about 43.2% of the fresh weight or 28.7% of the dry matter weight of the whole coffee fruit The fiber content (including cellulose, hemicellulose and lignin) in coffee pulp accounts for 51% (compared to dry weight), other components such as protein, pectin, starch, reducing sugar, accounts 49 % Coffee pulp is considered to be a very good food source for animals because of its relatively high nutrient content However, the coffee pulp contains some non-nutritious ingredients that are not good for metabolizing food for animals such as tannins or caffeine Besides, the coffee pulp also contain a large amount of fiber: 25.88% cellulose; 3.6% hemicellulose and 20.07% lignin Therefore, coffee pulp is also considered as a source of lignocellulose biomass used in bioethanol production (2nd generation ethanol production) 2.2 Introduction to the pretreatment process Pretreatment is considered to be the first and foremost step of the ethanol production process from lignocellulose biomass because through the pretreatment process, the carbohydrate polymers in lignocellulose biomass are converted into simpler sugars than before when performing the fermentation process This conversion is usually done by enzymatic hydrolysis However, due to the heterogeneous and complex hignocellulose biomass (for example: cellulose crystallinity, degree of polymerization, moisture, surface area, lignin and hemicellulose binding levels), it is necessary to requires pretreatment in order to regulate enzymatic hydrolysis It is important for the pretreatment process to reduce lignin and hemicellulose as much as possible in the material while retaining as much cellulose as possible and reducing the crystallinity of cellulose To evaluate the effectiveness of pretreatment, it is necessary to rely on the amount of reducing sugar released after hydrolysis as well as to consider the neutralization of pretreatment Besides, it is also important to consider the economics of the entire process when considering which pretreatment method to use 2.3 Introduction to the process of hydrolysis of coffee pulp The process of hydrolysis of coffee pulp after pretreatment is the biological resolution process of cellulose and hemicellulose in coffee pulp under the effect of enzymes Usually, cellulose will hydrolyze to form glucose, which is similar to the process of hydrolyzing starches into glucose But the difference is that glucose in cellulose is connected to beta bonds in the crystal structure, which is much harder to decompose than alpha bonds in amorphous starches (Holtzapple, 1993) In order to completely hydrolyze cellulose into glucose, it is necessary to catalyze the coordination of a cellulase complex Three types of enzymes are involved in this complex: endo-β-1,4-glucanases, exo-β-1,4-glucanases and và-glucosidases Hemicellulose can be hydrolyzed by enzymes such as endoxylanases, exoglycanases, xylanases, endomannanases, x-xylosidase, etc Besides, the dilute acid and alkaline agent can hydrolyze hemicellulose 2.4 Researches on pretreatment and ethanol production In recent decades, there have been many studies on the pretreatment and production of ethanol from lignocellulose biomass such as straw, wheat, bagasse, corn stalks, corn cob, grass or soft wood There are only a few studies on bioethanol production from coffee pulp Most recently, according to research (Shenoy et al., 2011) investigated the process of ethanol production from coffee pulp by hydrolysis method by 2% H2SO4 acid for 30 minutes at 90°C The total sugar content obtained after hydrolysis is 1.62 g/100 mL of hydrolyzate, the reducing sugar is 0.7 g/100 mL of hydrolyzate Then fermented and obtained ethanol amount of 0.46 g/g sugar Another study was also conducted on the object is coffee husk Coffee husks are hydrolyzed with to 5% H2SO4 acid, the material ratio: acid is 1:10 and the hydrolysis time is hours The hydrolyzate mixture was then fermented with commercial yeast S cereviciae at 30°C, pH = 5, within 24 hours, the highest ethanol concentration was 7.9 g/L ( Sahu, 2014) Most previous studies have focused on using H2SO4 acid in medium to high concentrations for pretreatment combined with lignocellulose biomass hydrolyzate to collect reducing sugars, the main advantage is the treatment time greatly shortened compared to using enzymes to hydrolyze In addition, economics is a factor that is always considered when conducting commercial production and previous studies meet this because if using enzymes for hydrolysis, it will be much more expensive than using acids However, the major drawback when using acid for hydrolysis is causing severe environmental pollution, the device requires to withstand acid and high temperatures Therefore, the latter studies hardly use acid to hydrolyze but use enzymes According to research on the topic "Technology research on processing some agricultural by-products with high pressure water to collect ethanol solution of fermentable sugar" (Nguyen Hoang Dung, 2008), agricultural by-products Industry used is straw, rice husk To make ethanol, straw, rice husks are treated by a hydrothermal reactor on a laboratory scale The research was then continued on a pilot scale on highpressure steam supply equipment This hydrothermal equipment is provided by Tokyo University (Japan) 2005, the research team is led by Dr Phan Dinh Tuan, Ho Chi Minh City University of Technology studied researching of technology to handle waste products in agricultural production such as straw, rice husk, rice husk in order to produce bioethanol and proceed to build a "biomass town" model in Thai My commune, Cu Chi district, Ho Chi Minh City After nearly years of implementation, scientists have successfully researched and produced biofuel from straw and cellulose-based waste (Hong Hoa, 2014) However, one of the difficulties of the project is the high cost of biofuel produced from straw, due to the high cost of cellulose decomposition in straw This process consumes a lot of energy, efficiency and low sugar concentration, resulting in much lower fermentation efficiency and ethanol concentration than starch ethanol production If decomposing cellulose with chemicals, the cost will be lower, but the resulting sugar solution will also contain chemicals that are not conducive to ethanol fermentation Therefore, this project aims to not use chemicals to break down cellulose The cost of refining ethanol after fermentation is also a difficult, costly energy issue, despite success in producing biofuel from straw, but to commercialize the product, scientists is continuing research efforts to lower production costs There were also many studies on the dilute alkaline pretreatment on meterials such as straw, bagasse, wood, For each type of material, there must be a separate survey to find out the optimal pretreatment method Most conventional agricultural biomass types had optimal pretreatment regimes However, on coffee pulps, there were not many in-depth studies The major disadvantage of using alkali for pretreatment was the difficulty in recovering the amount of alkaline in the hydrolysis solution In addition, the use of chemicals requires equipment with high temperature resistance and corrosion resistance This method was not recommended for use on an industrial scale because of its relatively serious potential for environmental pollution CHAPTER MATERIALS AND METHODS 3.1 Materials and methodology 3.1.1 Materials Robusta coffee pulp was collected at Pong Drang commune, Krong Buk district, Dak Lak province, Vietnam The berries were of bright-red colour, ripe, neither crushed nor moldy After harvesting, the pulp was removed and dried at 65°C until the moisture content was 5÷8% After that, the pulp was crushed and sieved; the diameter of the powder was 0.5÷1 mm Finally, the powder was packaged in plastic bags and stored under ambient conditions 3.1.2 Enzyme and yeast The enzymes use: Viscozyme® Cassava C: (Bagsvaerd, Denmark), Celluclast® 1.5L (Novozyme, Denmark), Glucosidase (Novozyme, Denmark) Yeast: Sacharomyces cerevisiae 3.1.3 Study objects methods Pretreatment method Dried coffee pulp (50 g) was treated by 500 mL of H2SO4 2% (w/w) solution in autoclave at 140oC for 45 minutes Taken out to cool at room temperature and then the pretreated biomass was recovered by filtration and washed by water After that, added 500 mL of sodium hydroxide solution (0.2 g NaOH/g biomass) and then the mixture was pretreated at 120oC for 20 mins then transferred to the microwave to pretreatment at 327 W, 20 minutes in the microwave system Finally, the pretreated residue was pressed to remove excess water and dried at 65oC until moisture content obtain from 5÷8% (Phuong, Le Pham and Le Nguyen, 2019) Hydrolysis method (enzyme loading) Amount of mL of enzyme cellulase preparation, 150 mL of 0.05 mol/L citrate buffer (pH 4.8), and 15 g (equivalent to 10% of dry material per 100 mL of solution, w/v) of pressed pretreated dried pulp were mixed in flask The containers were incubated in a thermal shaker at 50oC, 150 rpm for 72 hours and then the material from each treatment was centrifuged at 2,500 rpm during 10 mins The supernatant was removed for determination of RSs, total reducing sugars (TRSs), and glucose concentrations (Phuong, Le Pham and Le Nguyen, 2019) Fermentation method The solution after hydrolysis process was divided into equal portions of 250 mL each and taken in the erlen flask Then the solution was added by (NH4)2SO4 (1 g/L), K2HPO4 (0.1 g/L) and MgSO4.7H2O (0.2 g/L); the medium was autoclaved at 121oC for 20 mins and then cooled at the room temperature Fermentation was carried out in the erlen flask with 3x108 cfu/mL of S cereviciae at incubation temperature of 30°C at 120 rpm and pH of (Phuong, Le Pham and Le Nguyen, 2019) Effect factors: Strains of mold were identified in exp Criteria for evaluation: Các dòng nấm mốc có khả tổng hợp cellulase cao dựa đường kính vịng phân giải CMC Exp 9-15: Effect of culture media conditions on the ability of celulase enzyme biosynthesis Effect factors: Incubation temperature, incubation time, pH, potato extract concentration, substrate, additional mineral, additional nitrogen Criteria for evaluation: Cellulase enzyme activity (CMCase) Exp 16-19: Effect of concentration of precipitating agent (NH4)2SO4, NaCl, ethanol and acetone on cellulase enzyme activity Effect factors: (NH4)2SO4 concentration, NaCl concentration, tỷ lệ ethanol/enzyme ratio, acetone/enzyme ratio Criteria for evaluation: Activity, specific activity, recovery yield 3.2.4 3rd content: Investigate the hydrolysing process Exp 20: Effect of enzyme type on reducing sugar content obtained after hydrolysis Effect factors: Crude enzyme, Viscozyme, Celluclast 1.5L, Glucosidase Criteria for evaluation: Reducing sugar, total sugar Exp 21-22: Effect of enzyme concentration, temperature and time on reducing sugar content obtained after hydrolysis Effect factors: Enzyme concentration, hydrolysing temperature, hydrolysing time Criteria for evaluation: Reducing sugar, glucose Exp 23: Optimization of hydrolysing process Effect factors: Enzyme concentration, hydrolysing temperature, hydrolysing time Criteria for evaluation: Reducing sugar 3.2.5 4th content: Exp 24: Effect of Ca(OH)2 volume on hydrolytic detoxification efficiency Effect factors: Ca(OH)2 volume Criteria for evaluation: Precipitation weight 3.2.6 5th content: Investigate the fermentation process Exp 25-27: Effect of yeast cell density on the content of ethanol formed after fermentation Effect factors: Yeast cell density, fermentation temperature, fermentation time Criteria for evaluation: Ethanol content 11 Exp 28: Effect of fermentation method on ethanol content obtained Effect factors: SHF, SSF, SHF + SSF Criteria for evaluation: Ethanol content CHAPTER RESULTS AND DISCUSSION 4.1 Determining the chemical composition of the Robusta coffee pulp The results show that the chemical composition of coffee pulp includes: Total sugar 9.18±0.23%, pectin 4.37±0.06%, protein 9.52±0.23%, cellulose 25.88±0.91%, hemicellulose 3.6±0.18%, lignin 20.07±0.56%, lipid 1.22±0.11%, caffeine 0.78±0.01%, polyphenol 8.69±0.12% and ash 6.29±0.09% 4.2 Effect of pretreatment methods on changes in cellulose, hemicellulose and lignin content 4.2.1 Effect of the extraction method of decaffeine and depolyphenol efficiency For maceration method: Decaffeine efficiency was 88.1% and depolyphenols was 80.6% For ultrasound-assisted extraction method: Decaffeine efficiency was 91.4% and depolyphenols was 85.5% For microwave-assisted extraction method: Decaffeine efficiency was 92.3% and depolyphenols was 87.7% Method of caffeine and polyphenols was chosen is maceration because of its economy, efficiency and convenience 4.2.2 Optimize the process parameters of decaffeine and depolyphenol In order to find the optimal value in decaffeine and depolyphenol with hot water, as well as the interaction effects of factors on the caffeine and polyphenol efficiency, The optimization of hot water extraction was performed by response surface methodology with parameters changed: X1: Solvent/material ratio, X2: Extraction temperature, X3: Extraction time and two desirable responses such as Y1 (%, decaffeine efficiency), Y2 (%, depolyphenol efficiency) (Table 4.1) Table 4.1: Coded level and actual values of independent variables Independent variables Symbols Solvent/material ratio (v/w) Extraction temperature (oC) Extraction time (phút) X1 X2 X3 12 -1 30/1 70 90 Coded level 40/1 80 120 50/1 90 150 The regression equations were shown below: Y1= 88.33–3.56X1+5.17X2–7.6X12–9.97X22–5.23X32+4X1X3+4.49X2X3 (1) Y2= 80.65–1.28X1+1.59X2–4.24X12–3.19X22+0.52X1X2+0.77X1X3 (2) The good fits of the regression equations were achieved and the analysis of variance (Anova) was statistically significant (p0.8) and Q2 (0.968 and 0.914, >0.5) value are high Hence, this was the robust statistical model Table 4.2: Results of the optimal model Results Optimal value Experimental value X1 38.6/1 X2 82.9 X3 136.7 Y1 88.1 89.52±0.49 Y2 80.95 78.77±0.44 The optimal results obtained from the initial selection model were: decaffeine efficiency reaches 89.52% and depolyphenol 78.77% at the condition that the solvent/material ratio was 38.6/1, the extraction tempature was 82.9oC and the extraction time was 136.7 minutes 4.2.3 Effect of acid H2SO4 pretreatment on changes in cellulose, hemicellulose and lignin content As the acid concentration, temperature and pretreatment time increased, the decrease in cellulose, hemicellulose and lignin increased However, dilute acids are actually effective in eliminating hemicellulose but not highly effective for lignin Specifically, after pretreatment with dilute acid, cellulose lost 7.5%, hemicellulose was removed 43.9% and lignin removed 4.2% The optimal pretreatment condition with dilute H2SO4 acid for coffee pulp were: H2SO4 2% (w/w), acid:material ratio was 10:1, temperature pretreatment 140oC and time was 45 minutes A–B Figure 4.1: The surface of coffee pulp before (A) and after (B) pretreatment by H2SO4 2% under scanning electron microscopy (SEM) 13 4.2.4 Effect of alkali NaOH pretreatment on changes in cellulose, hemicellulose and lignin content Dilute alkali pretreatment was actually more effective than acid when removing 46.11% hemicellulose, 76.63% lignin from raw materials but retaining 71.25% cellulose Although this result is better than pretreatment with dilute acid, it was not the best The optimal pretreatment condition with NaOH dilute alkali for coffee pulp were: Alkali ratio of 0.2 g/g material, material/alkali ratio was 1:10, pretreatment temperature of 120oC and time was 20 minutes A–C Figure 4.2: The surface of coffee pulp before (A) and after (C) pretreatment by NaOH 0.2g/g under scanning electron microscopy (SEM) 4.2.5 Pretreatment of coffee pulp with P chrysosporium strain After 50 days of pretreatment: cellulose decreased by 23.7%, hemicellulose decreased by 14.1% and lignin decreased by 51.4% The pretreatment method of P.chrysosporium was not really effective in removing hemicellulose and lignin However, this method was performed at room temperature and did not use chemicals, so it was low cost and did not pollute the environment A–D Figure 4.3: The surface of coffee pulp before (A) and after (D) pretreatment by P chrysosporium under scanning electron microscopy (SEM) 14 % removed 4.2.6 The effect of combining many pretreatment methods on coffee pulp 100 90 80 70 60 50 40 30 20 10 Cellulose 76.6 Hemicellulose Lignin 78.5 68.7 54.8 43.9 47.1 28.7 34.2 79.2 71.4 77.4 50.2 34.4 29.6 20.7 12.8 7.5 4.2 Pretreatment methods Figure 4.4: The change in fiber content by different pretreatment methods There has been a significant improvement in the percentage of elimination of fiber content in the coffee pulp when there is a combination of or pretreatment methods together In terms of elimination of hemicellulose and lignin as much as possible, the acid-alkalinemicrofiltration pretreatment method was chosen, when 71.4% of hemicellulose and 79.2% of lignin were removed However, considering the least cellulose loss, the acid pretreatment method was chosen, when the loss was only 7.5% of cellulose A–E Figure 4.5: The surface of coffee pulp before (A) and after (E) pretreatment by acid-alkali-micro wave under scanning electron microscopy (SEM) Therefore, to have a basis for choosing which pretreatment method is best, it is necessary to consider how the reducing sugars created after the 15 hydrolysis process of these methods will change For the reduction path objective function generated at the highest pretreatment method, that method will be selected Table 4.3: Influence of different pretreatment methods on the formation of reducing sugars Pretreatment methods Reducing sugar g/L Acid Alkali Micro AcidAlkali Alkaliacid Alkalimicrowave 19.85 28.21 13.19 41.27 39.29 38.21 Acidalkalimcrowave 43.26 The results of Table 4.3 show that there was a basis for selecting an appropriate pretreatment method for coffee hulls that is a combination of acid-alkali-microwave Hemi Ash 1.05 5.20 % % Celulose 25.88% Other 44.09% Lignin 20.07% Ash Hemi 6.29% 3.67% Other 12.09% Celulose 77.49% Lignin 4.17% Pretreatment with acidalkali-microwave Non pretreatment Figure 4.6: Ingredients of materials before and after pretreatment The remaining prepretreatment materials were mainly cellulose (77.49%, based on the initial cellulose content), ash and some other substances (Figure 4.6) The pretreated coffee pulp have sensory characteristics that are soft, light yellow-brown and have good water-repellency 4.3 Recovery cellulase enzyme from mold 4.3.1 Isolation of some molds strains with morphological characteristics like Aspergillus sp and Trichoderma sp Bảng 4.4: The mold strains were isolated from a number of sources No Source Coffee land Coffee pod Rotten leaves Tatal Number of Aspergillus strains 13 Symbols DA1, DA2 QA1÷QA6 CA1÷CA5 16 Number of Trichoderma strains 11 Symbols DT1, DT2 QT1÷QT5 CT1÷CT4 Through preliminary observations of the macro and microscopic characteristics can be confirmed, among 24 surveyed strains, there were 13 strains with Aspergillus like characteristics and 11 strains with Trichoderma like characteristics 4.3.2 Investigate the cellulase biosynthesis of isolated mold strains Of the 24 isolates, the group of molds isolated from pods and branches exhibited higher cellulase activity than coffee land Particularly, 11 strains isolated from pods have strains for high activity (QA1, QA3, QA5, QT4, QT5) With strains isolated from leaves, strains showed high cellulase activity (CA1, CT2, CT4) The remaining strains were isolated from land but none showed high cellulase activity (D 0.5) value are high Hence, this was the robust statistical model Table 4.8: Results of the optimal model Results Optimal value Experimental value X1 X2 X3 Y 24.77 51.19 92.3 27.35 26.22±0.39 Optimal results obtained from the initial selection model were: Reducing sugar content reaches 27.35 g/L at conditions of enzyme concentration of 24.77 FPU/g, hydrolysis temperature of 51.19oC and time hydrolysis was 92.3 hours 4.4.5 Research on hydrolytic detoxification process The acid-alkaline-microwave pretreatment process produces a number of potentially toxic substances for yeasts, that were HMF and Furfural, with a content of 2.11 g/L HMF and 3.37 g/L furfural The method to remove some of these substances was the calcification method developed by Millati (Millati et al., 2002) in combination with activated carbon filtration Precipitation weight (mg) 0.5 10 0.4 pH 0.3 0.2 0.1 0 Ca(OH)2 Volume (mL) Precipitation quntity (g) 11 Figure 4.7: Influence of Ca(OH)2 volume on precipitation formation At pH 10.2, the amount of precipitate formed peaks and tends to decrease with increasing pH However, it can be noticed that the sulphate radicals are precipitated almost completely at pH value of 9.8 21 4.5 Research on fermentation hydrolyzate 4.5.1 Effect of initial yeast cell density added to fermentation When the yeast cell density increased from 3x106 to 5x108 cfu/mL, the ethanol content also increased However, the increase was not significant If the amount of yeast continues to increase, the content of ethanol tends to decrease The reason is that with the high density of yeast cells, yeasts will use the sugars formed after hydrolysis to increase biomass, so the ethanol content will not increase any more but will decrease over time Accordingly, an additional yeast cell density of 5x107÷3x108 cfu/mL was selected for further experiments 4.5.2 Effect of fermentation temperature on the content of ethanol S.receive yeast is resistant to heat and fermentation at 42oC However, according to this study, the yeast can ferment well at temperatures of 30÷40oC If the temperature increases to 45°C, the fermentation efficiency decreases sharply and at 50oC the yeast almost does not grow and ferment Ethanol content (g/L) 12 10 9.91 9.05 8.66 8.11 10.1 9.16 8.57 5.02 7.11 6.22 3.71 5.79 4.44 24 h 6.02 48 h 4.12 1.8 4.25 3.12 45 50 72 h 25 30 35 40 Fermentation temperature (oC) Hình 4.8: The influence of fermentation temperature on the content of ethanol obtained Although the temperature of 35oC has proven to be the best for fermentation, when considering other factors such as cost and equipment, the selected fermentation temperature is 30oC 4.5.3 Effect of fermentation time on the content of ethanol obtained Analysis results show that the content of ethanol generated during fermentation increases with time of fermentation and reaches a maximum at 72 hours of fermentation with 72.55% After 72 hours, the ethanol content did not increase and tended to decrease slightly 22 Ethanol content (g/L) 12 10.06 10 8.41 6.71 9.02 9.46 9.25 6.07 4.88 4.39 3.25 24 Glucose content (g/L) After 72 hours of fermentation, the fermentation efficiency reaches 75.96% which means that 75.96% of the glucose is converted into ethanol by yeast Ethanol Glucose 48 72 96 120 Fermentation time (hour) Figure 4.9: The influence of fermentation time on the content of ethanol obtained Analysis results Figure 4.9, show that the fermentation condition at 30oC, with an initial cell density of 3x108 cfu/mL and fermentation for 72 hours was suitable when the ethanol content and fermentation efficiency are the highest 4.5.4 Effect of fermentation method on the content of ethanol obtained Table 4.9: Comparing fermentation SHF, SSF SHF+SSF Hydrolysis time (day) Fermentation time (day) Hydrolysis/ fermentation temp (oC) 50/37 50/37 50/37 50/37 50/37 50/37 Ethanol (g/L) SHF SSF 5,34±0,18a 4,76±0,14a 9,02±0,1a 11,28±0,19a 11,38±0,21a 10,29±0,13ab 7,1±0,12b 8,13±0,14b 10,09±0,08b 10,79±0,15b 10,72±0,11a 10,44±0,13a SHF+SSF 8,96±0,09a 9,43±0,16c 10,08±0,13c 10,23±0,16b 10,26±0,06a Based on the research results, if not considering the time, the fermentation method SHF obtained higher ethanol content than fermentation SSF and SHF + SSF Specifically, at days, the ethanol obtained from SHF fermentation was 11.28 g/L, 10.5% and 16.4% higher than that of SSF and SHF + SSF However, considering the energy and implementation costs, the SHF method is more expensive than the other two methods 23 Besides, the fermentation of SSF and SHF + SSF gives the same results but if applied on an industrial scale, SHF + SSF fermentation allows to increase the concentration of dry substrate significantly, by performing hydrolysis first by enzymes to dilute the fermentation fluid, then proceed to add coffee hulls and ferment SSF CHAPTER CONCLUSION AND SUGGESTION 5.1 Conclusion The pretreatment combination of acid-alkaline-microfiltration gives the best treatment effect This combined method removes 71.4% of hemicellulose and 79.2% of lignin from the material but retains 69.5% of cellulose from the original The best way to get rid of caffeine and polyphenols is to use 90°C water to extract for 120 minutes Decaffeine and depolyphenol is done after pretreatment will save a certain amount of water A strain of Trichoderma asperellum (QT5), was selected, isolated from damaged coffee berries Cellulase enzyme obtained from this fungal strain after preliminary purification activity reached 21.72 CMCase/mL and 43 FPU/mL Cellulase* enzyme were applied in the hydrolysis and fermentation of coffee pulp to bring a relative effect Although the reduced sugar and glucose content of cellulase* (in combination with Glucosidase) was lower than when using commercial enzymes (Viscozyme+Glucosidase), the ethanol content was only lower 13.8% Proposing a solution to eliminate the toxins for yeast that is using Ca(OH)2 to adjust to pH = 9.8 will make the most of the Furfural and HMF After six days of hydrolysis and fermentation, fermentation SHF was higher efficiency than SSF or SHF+SSF when the ethanol content reached 11.28 g/L This content was higher than fermentation methods SSF and SHF+SSF of 7.4% and 9%, respectively 5.2 Suggestion In order to improve the efficiency of the pretreatment process as well as the hydrolysis process, the reducing sugars should be recovered from the pretreatment process to add back to the fermentation process or separate fermentation In order to improve the efficiency of hydrolysis by cellulase enzyme, the next study should study the mechanism of cellulase enzyme absorption on cellulose molecule surface This absorption process can be increased by 24 adding some surfactants such as Pluronic F68, Polyoxyethyleneglycol, Emulgen 147, Neopelex F-25 or Cationic Q-86W In order to further reduce the costs of hydrolysis and fermentation processes, the further study should find ways to effectively use certain immobilizing enzymes and other immobilizing yeasts 25 ... CMCase activity of 1.6 U/mL Effect of environmental pH and concentration of potato extract on CMCase activity The CMCase activity increases with increasing environmental pH and maximum activity at... Library of Vietnam LIST OF PUBLISHED WORKS Do Viet Phuong, Pham Van Tan and Le Nguyen Doan Duy 2017 Determination of caffeine in coffee pulp (Coffea robusta) using UVVisible spectrophotometer Vietnam... pretreatment process and the microbiota for coffee pulp (Coffea robusta) hydrolysis to produce ethanol" 1.2 Objective of the study Using acid, alkali, microwave or white rot fungi to pretreatment