MINISTRY OF EDUCATION AND TRAINING NONG LAM UNIVERSITY - HO CHI MINH CITY Faculty of Chemical Engineering and Food Technology PRODUCTION OF HIGH-VALUE CHEMICALS FROM CORNCOBS BY CANDIDA MAGNOLIAE TISTR 5664 A Thesis submitted in partial fulfilment of the requirements for admission to the degree of Bachelor of Engineering in Food Technology By Student: Tran Ngoc Bich Hong Supervisor: Assoc. Noppol Leksawasdi Dr. Duong Thi Ngoc Diep Ho Chi Minh City, 2024 MINISTRY OF EDUCATION AND TRAINING NONG LAM UNIVERSITY - HO CHI MINH CITY Faculty of Chemical Engineering and Food Technology PRODUCTION OF HIGH-VALUE CHEMICALS FROM CORNCOBS BY CANDIDA MAGNOLIAE TISTR 5664 A Thesis submitted in partial fulfilment of the requirements for admission to the degree of Bachelor of Engineering in Food Technology By Student: Tran Ngoc Bich Hong Supervisor: Assoc. Noppol Leksawasdi Dr.
Duong Thi Ngoc Diep Ho Chi Minh City, 2024 ACKNOWLEDGMENT This project took a great deal of labour, help, and support from many people and organizations to be completed. Initially, regarding the internships, Nong Lam University, Ho Chi Minh City, and Chiang Mai University collaborated. I would like to sincerely thank the faculties of Chemical Engineering and Food Technology at Nong Lam University and Agro-Industry at Chiang Mai University for providing me with the invaluable opportunity to take part in a four-month internship program. I would especially want to thank my advisor, Associate Professor Dr.
Noppol Leksawasd1, for the opportunity, excellent support, and direction he provided me with throughout my four months of study. Also giving my gratitude towards my co-advisor, Ms. Su Lwin Htike, Mr. Sumeth Sommanee, Ms.
Chatchadaporn Mahakuntha, Ms. Kritsadaporn Porninta and Ms. Juan Feng, for their valuable guidance, advice, and remarks, which have enabled me to practice and conduct research at the Bio-research Cluster, Food Engineering Division, Faculty of Agro-Industry, School of Agro-Industry, Chiang Mai University. I want to thank my friends and lab partners for all of their support during my time in the lab.
I also want to thanks Dr. Duong Thi Ngoc Diep for her advice, encouragement, and assistance in my report. Lastly, I would want to express my sincere gratitude and love to my family for their acceptance and understanding of my hard work throughout my internship. Without the assistance I got from everyone, I could not have finished my studies.
Tran Ngoc Bich Hong Ho Chi Minh City, May 20, 2024 ABSTRACT PRODUCTION OF HIGH-VALUE CHEMICALS FROM CORNCOBS BY CANDIDA MAGNOLIAE TISTR 5664 Hong Tran Ngoc Bich, Noppol Leksawasdi, Diep Thi Ngoc Duong Faculty of Chemical Engineering and Food Science Technology, Nong Lam University — Ho Chi Minh city Email: 19125515@st.vn Corncobs, a by-product of maize production, are being known as a low-cost feedstock for high-value chemicals production. This research studied on the potential for producing high-value chemicals including xylitol, ethanol and phenylacetylcarbinol (PAC) from corncobs. Here, hemicellulosic and cellulosic hydrolysates of both original and concentrated forms were being compared in the production of xylitol and ethanol, respectively. The original hydrolysates were concentrated by vacuum evaporation at 70°C to achieve a xylose and glucose concentration of approximately 100g/L for cultivation using Candida magnoliae TISTR 5664.
In regard to xylitol production, the xylitol yield obtained from concentrated hydrolysates (97.18 g/L of xylose) with a value of 0.79 g/g, was statistically significantly higher (p < 0.05) than the original hydrolysate (21.24 g/L of xylose) with the xylitol yield of 0. For ethanol production, the xylitol yield obtained from the concentrated hydrolysates (99.08 g/L of glucose) was statistically significantly higher (p < 0.05) than that of the original hydrolysates (38.22 g/L of glucose) with values of 0. Moreover, the collected frozen-thawed whole cells obtained after cultivation were used as a biocatalyst in a two-phase emulsion system, applying two different phase volume ratios of buffer and organic solvent (1:1 and 1:0:43) for PAC biotransformation. According to the results, the PAC concentration from 1:0.43 phase volume ratio (7.28 mM) was statistically significantly higher than that from the 1:1 phase volume ration with a value of 5.
These findings suggest corn cobs as a valuable il feedstock for the co-production of high-value chemicals, offering a sustainable and cost-effective solution for the biorefinery industry. Keywords: Corncob, Candida magnoliae TISTR 5664, xylitol, ethanol, phenylacetylcarbinol 1H TABLE OF CONTENTS ACKNOWLEDGMETITT.- 5 << SH HH HH HH ng i ABS TRAC Pos ccsssscanscas ss swesnsnsnnsowastanesbusstashun scowaseawete xesnssesasinas cuaaasounsvessanaceunceaactaiozesteees ii TABLE OF CONTENTS wisssssccessssscssnssesssscssssusessnsncavsscserecssesssnsssessvsnsssecssnsseeosassssevess iv LESTE, GE TABLES ccscessrnsersseseneseossenseneennnnsronsceaneanenssnsossnsnsevasananuneesensnsensvenseeresseeseneanen ix LISTE OB BIGURES sesescsccccset cosscteccvscssstessnscdeasessscecsszscoensoasssesteasensaceesseesesessesszenenases xii LIST. OF ABBREVIATIONS 2060620256105 15811615013 635550535884EKS845586544865E3Ep 4e Xv CHAPTER 1 INTRODUCTTON. ¿c0 n0 ng ngà nề H1 K14 465884554 065666 1 ID 006.
| IVUAAN Cu) cua. 3 CHAPTER 2 LITERATURE REV[EYW. HH“ HH HH HH g4 re 4 2.1 Introduction Of COmCODS sssccssssssccibiisidanio gia 0181811183618 00x08143.458110348636863615113613 65988 4 DEAD lia) shugE5900PEEERGIELEHBHSEEEEHHESESISEDHESIDiHEHGBEERISTENIESHRGEĐ-HHEGS-EONSĐSSBSHSEAHDBESUiSiEDDRSVESS/02/UB- 6 2.1 Overview & applications of XyÏIfOÌ.ÄYLOL:DEOdUGLIOH ssxsgzssccsi162166621515562558055690638S638A38G8956 49885808 438)934 2EEUSE5EG348E83 68 8 DB, EANAIG syssssesseseg0i62409091E10913BSS'G/SEHGSBSHENGSHĐSSSLIEESGSISBESIUSBSIGIEGMESESMXG4D8G8399182008:9083143853 10 2.1 Overview and applications of ethanolL.Prơducffon:0f elhan0ÏLizssszssexzsesssssseevsesrssitxS2108548L83551950045E05348248g946 3554380685646 12 ZA Phenylacetylearbinol (PÀG]:ecorsasrdeesaodiesaktoigsssssgd42134661103053308550305308166013008510 066 14 2.1 Overview and applications of phenylacetylcarbinol.2 Production of phenylacetylcarbinol .5 Candida species — Candida magnoliae TISTR 5664.6 Overview of the transformation from lignocellulosic materials to xylitol, ethanol and phenylacetylcarbinol through fermentation using Candida magnoliae TISTR 5664 17 2:6.PTelrediifGrlil,TIGGESŠ bscsesetoattidlkbisdobibagtgf4SESD3S8048GESuSEE53ug/80900108E13G8EE14SGGE.Enzymatic Wy dro ly 819 toseeissstssttssitbi806868g6Ei000ui08ã68u)0ã0guj2g300346i2x)38-g3AggagBdEgiCGiibgge4 21 2.3 Biotransformation of high-value chemicals .-- --------- --<+5-<+55 22 CHAPTER 3 MATERIAL AND METHODOLOGY.1 Timevand place GETeseatCh: ác ssesessssssssssssszasssEeseAdle4468,21116u0060l6e38d00.2 Materials, chemicals and equipment .-- ----- -- +52 + +52 +£++*£++£<+e<+zeezzeeze 24 B21 Material cssssesesssaessosssatisss36tsgsgiaS5 tùnh3S5348060880.55000580480005 0300 0116808%1045005003001458000E00588 24 Sun Lai: (CGTPIGDDN+spibrsauniitioliktttitteiipsotiSgBEEHIGERIIEPEIMEDHPIESSHDTHHISGBDDTISEDSEHIEISDIBEERGGRPEIS 24 3.2 Candida magnoliae TISTR Š6644. ES Site 25 50/0): (COHGTHITLGỗISbssssosngiEntuietiiosodbeintsstibSoigid50Ghgqisudisggu3i38i4645438.3 EQUIPIM Gt bss ccncssesnssenencnsscssewessossnseenceeetet seve aoe unesauennssarerneareememmrennascess 26 SOU Ch 1Q ƯỒ ———— aa ee 26 3.1 Dilute acid pretreatment «2.2 Enzymatic hydrolysis on corncob cellulosic residue.3 Vacuum evaporation PrOcess.4 Microorganism and inoculum preparation .5 Cultivation process for xylitol and ethanol production.6 Selection of frozen-thawed whole cells for PAC biotransformation .7 Biotransformation process to produce PAC using frozen-thawed whole cells E80 (vn 0.9 Atialytical Tii€tHGŠ isncsc.1 Determination of sugars, xylitol and ethanol concenfrafions.2 Determination of dried biomass COHNC€HfTđfÏOH.3 Calculation ofyields (Yojtte, ctl, ¥ Vals) sasssasscnisssssnsoraceasanssioeteaveasincanteavans 36 3.4 Determination of Volumetric PDC Activity .5 Determination of PAC, pyruvate, benzaldehyde and benzoic acid COMECTUUF OL ONS ccsseren satya erecta eta ig SSS Se SELON NOS oe SE STOUR EIT ELANCE SESS 38 3.6 Determination of pyruvate molarity balance and benzaldehyde molarity Coe SE ee ee ee ee ee ee ee ee ee can co 40 3.10 Statistical anaÌyS1S.
nHH HH HH HH ngư 40 Chapter 4 RESULTS AND DISCUSSION.1 Xylitol production from hydrolysate after dilute acid pretreatment .1 Effect of sugar concentration on the formation of xylitol and biomass production in dilute acid pretreatment .2 The changes of pH and total soluble solid (°Brix) in xylitol production.2 Ethanol production from enzymatic hydroÌySafes.1 Effect of sugar concentration on the formation of ethanol and biomass production in enzymatic hydrolysis .2 The changes of pH and total soluble solid (°Brix) in ethanol production.3 Phenylacetylcarbinol biotransformation using frozen-thawed whole cells.1 Selection of frozen-thawed whole cells for biotransformation process.2 Effect of volumetric PDC activity, pyruvate and benzaldehyde on the biotransformation process of PAC in two-phase emulsion system. 56 Chapter 5 CONCLUSIONS AND RECOMMENDATIONS. RECOMMENDA ONS ieee eaten carer ee ae ee 62 REE ERENCES coi s5 HE 04133 GH290.1513E1ÄNESAGESESSSEAESSSSSSGS03385598450239904688 64 Ä PPENDIC E2 cg6 ác 01xEngg46123568X6336E301S6633500sssxduioke4gzLg8d3gusnhisszs tuagiiStuscanyde 72 Ve] [eh 0 00, 2 See ernetnern reteset etre eran cere tere ern eer tere rena rete es 72 A.1 Standard curve for analyzing sugars, xylitol, and ethanol concentrations.2 Standard curve for analyzing PAC, benzaldehyde (Bz), pyruvate (Pyr) and benzoic acid (Bzc) concentrations.0s8i0iBianlSgi0lg4BiG0g3i0.1 Analyze sugars, xylitol concentration and dried biomass concentration in hemicellulose hydrolysate .2 Analyze pH and total soluble solid in hemicellulosic hydrolysate .3 Analyze sugars, ethanol concentration and dried biomass concentration in hemicellulose] Hy drOly Sate s.4 Analyze pH and total soluble solid in cellulosic hydrolysate .5 Analyzed volumetric PDC activity, concentrations of PAC, pyruvate, benzaldehyde and benzoic acid in biotransformation process using frozen-thawed whole cells C.magnoliae TISTR 5664 as a biocatalyst .0 eee eee cece 90 B.6 Analyzed volumetric PDC activity, concentrations of PAC, pyruvate, benzaldehyde and benzoic acid in biotransformation process using frozen-thawed whole cells C.- 2c 2S reirey 91 AP PONCIK: C sesesssescasconsaennen aasinnseensyansce 5588861103055 813533618138880S8380800:45388.1 The result of the SPSS analysis in xylitol production with normal hemicellulosic //9x04711712212575 .2 The result of the SPSS analysis in xylitol production with concentrated Herfileellulösĩ6: Hy ALOLY SACS crc anscsnacsscsynseenam sere nomnsrasmesirsunmsiessasusnaateansureaumeusenencuma 101 C.3 The result of the SPSS analysis in ethanol production with normal cellulosic hydrolysates sis memes ERE eee EEN 105 C.4 The result of the SPSS analysis in ethanol production with concentrated cellulosic hydroÏysate.-- ----------~- i ttt ili ii 108 C.5 The result of the SPSS analysis in PAC production with two-phase emulsion System ratio Of Li).6 The result of the SPSS analysis in PAC production with two-phase emulsion System Patio OF 15043 sssccsess cones seusensxsssesemseresssamaneeneveaeeenus oe areca: 112 Vill LIST OF TABLES Table 3. Listof chemicals Used 1H.
this ĐFO|GCE cece reser cesssvoccasevensmernssrveressenenssmnnseees 25 Table 3.2 List of equipment used in this prOJ€CE.1 Effect of glucose, xylose and arabinose on the formation of xylitol and biomass production during the dilute acid pretreatment process of dilute corncobs with normal sugar concentration from 0-168h cultivation by C.2 Effect of glucose, xylose and arabinose on the formation of xylitol and biomass production during the dilute acid pretreatment process of dilute corncobs with concentrated sugar concentration from 0-168h cultivation by C. magnoliae TISTR 5664 Table 4.3 Dried biomass concentration (g/L) and production yield (Yxs) of hemicellulosic hyrdrolysate in normal and concentrated medium from 0-168h cultivation by C.cc:cceccceseceseceeeeeeeeeeeeeneceseesseeeneeeneeesees 44 Table 4.4 pH level and total soluble solid (°Brix) of normal and concentrated hemicellulosic hydrolysates from 0-168h cultivation by C.5 Effect of glucose and xylose on the formation of ethanol and biomass production during the enzymatic hydrolysis process of corncobs with normal sugar concentration from 0-168h cultivation by C.6 Effect of glucose and xylose on the formation of ethanol and biomass production during the enzymatic hydrolysis process of corncobs with concentrated sugar concentration from 0-168h cultivation by C.7 Dried biomass concentration (g/L) and production yield (Yx/s) of cellulosic hydrolysates in normal and concentrated medium from 0-168h cultivation by CMGSTONGE TIS TR. S604 esc nnseucsvsssreseevavarsnnsensacecesusnsaaunen yuesecemesunnesmmmareecneemnanenns 52 1X Table 4.8 pH level and total soluble solid (°Brix) of normal and concentrated cellulosic hydrolysates from 0-168h cultivation by C.9 Volumetric PDC activity of frozen-thawed whole cells obtained after 168 h cultivation by C. SH HS HH hy 55 Table 4.10 PAC production through biotransformation process in a two-phase emulsion system with a phase volume ratio of 1:1, using frozen-thawed whole cells of C.
magnoliae, TISTR 5664 aS :a.DìQGdEAÌWSL: ác can i22 cả tá hàng Hà ghê kgn 36g 0E58GLL46 .11 PAC production through biotransformation process in a two-phase emulsion system with a phase volume ratio of 1:0.43, using frozen-thawed whole cells of C. masnoliae TISTR 5664 a8 4, DiOCALALY 8 liscsnsesoncsenwmncucasa na 59 Table B.1: The concentration of glucose, xylose, arabinose from hemicellulose hydrolysate in original medium between cultivation 0 — 168 h.2: The concentration of glucose, xylose, arabinose from hemicellulose hydrolysate in concentrated medium between cultivation 0 — 168 h.3: The concentrations of dried biomass obtained in normal and concentrated hemicellulosic hydrolysate medium between cultivation 0 — 168 h.4: The data of pH and °Brix level in original and concentrated medium of hemicellulosic hydrolysates mediums between cultivation 0 — 168h.5 The concentration of glucose, xylose from cellulose hydrolysate in original medium between cultivation 0 — 168 h. --- -- cece cc S112 1111111111111 2 5111 11k x2 83 Table B.6 The concentration of glucose, xylose from cellulose hydrolysate in concentrated medium between cultivation 0 — 168 h.7: The concentrations of dried biomass obtained in normal and concentrated cellulosic hydrolysate medium between cultivation 0 — 168 h.8: The data of pH and °Brix level in original and concentrated medium of cellulosic hydrolysates mediums between cultivation 0 — 168 h.9: Volumetric PDC activity of frozen-thawed whole cells after 168 h of tữs¡0tnf3n11211151102727 57. ẻẽẽ ẽẽẽẽẽẽ 90 Table B.