Luận án tiến sĩ: Phát triển consortium vi sinh học xử lý lignocellulose - Vu Ngoc Ha Vi

I⁄I/^NIE HUNGARIAN UNIVERSITY OF AGRICULTURE AND LIFE SCIENCES Development of microbial consortium for biological pretreatment of lignocellulosic raw materials PhD. Thesis Vu Ngoc Ha Vi Budapest 2023 Doctoral School Name: Doctoral School of Food Science Field: Food Science Head: Prof. Livia Simon-Sarkadi D. Department of Nutrition Science Institution of Food Science and Technology Hungarian University of Agriculture and Life Sciences (MATE), Hungary Supervisor: Prof.

Department of Bioengineering and Alcoholic Drink Technology Institution of Food Science and Technology Hungarian University of Agriculture and Life Sciences (MATE), Hungary Prof. Vijai Kumar Gupta Ph. Biorefining and Advanced Materials Research Centre Scotland’s Rural College (SRUC), UK The applicant met the requirement of the regulations of the Hungarian University of Agriculture and Life Sciences and the thesis is accepted for the defense process. Signature of Head of Doctoral School Signature of Supervisors il TABLE OF CONTENT 1.

AND OUTLLIN Eivssicssnaissicsnnuneienisnsinenannsoaanbait tiviwnetowwsnsnadasoubsnnnaisetunaransswsatisn 1 lui: SU ole (CE (Cl) 1 L2. - TCSs ccaisoiczasoziaseinenn onnibivenscininrsinsiiuresaissnneivesiew tensa unnn tis leanne vanrdn ain lvaleidvaieaenansionlewiv 3 LITERATURE RIB VIEW sangtsstooititoagsies4LSSG. 4 Pilele TCE1TUlÖ§ TkzussoanotindoibitiDGEIGHHOIAGGEUOHROEHUSSNEIDSIGBDHIGNIOHRGIAGEAINGMRASESSAHERRiiNM 5 2:12: Hemice lulose ccavssscmemcemmensmanseaten See RIE UE ER 2) DAB. LAGI sznusioiiitpttineilEDiEIDESDSIEBIHEIBEERGEIIERHIEIRHEIREXEPSNSIEABEGEERIIEHSTHEIERGHĐSMESGPBiABBMM 6 2.

Pretreatment of lignocellulosic b1OImasSS.- ---- + 25232 *++E* + £*EEsereereerserrserree 7 22Qle PHYSICA Prewrea tiie t oc cececeemcerennersentnennrnee cermin tener 8 50; Chemica ppnetne altri en basics stesasssiiessnsisscisummntisnanssuision cmwciivansnnsiie tieduoacutinentlenbataaiiatmealanaets 9 22.3» -PHYsiOCheniical Preheat tit sccorcess seeeoeenrerreneayenvevenaennensncemmennnesmmusermmerrenveneneres 11 2.GÉbloetHanolpf6dl6HOi:sssssseeasenriniiesbitaodiiieibitigsbiBEEHUHOR-ISEEL469)023380368 21 SACCHATT EH CATION.-nnninenerssermnnnrannnnnernactinerpinscmnnns sienttaninsaiinwofsspnatoiesnensdnenneieauriirauinanidnrantigeneaneinannee see 21 AlCOHONIC fSFEIfIGIIEOTH ssssssessnerren cues 23958 8R4 140008356156 2Sh3ESE1EĐESGGAESSSS-EELRDHSAIRNGEESRORG-G14E4856051 22 MATERIALS AND METHOIDS. --- --- +21 2S S1 2x TH HT TH TH ng Hư 23 3.1, Lipnocellilose subSHáảÍEssesssssoeoasssrotrdoilstidbilioiigtsltKOREERSEHGEIHIRUĐBIESgSeSufe 23 3/2: - ,MIIGTGBTENHISHHBsswswpueeoiboeiiegoitdgioilotabeSAdS90LRQĐADiGBQOIAriSSLGAigaHgASGERJWANidtaibpszsrjNiOqesy 23 3. Effect of bacteria, yeast and their consortia on the pretreatment of lignocellulose. Fungal biolopical pretreatiientcc css cannnsmnmnsiasienuniovanmantnsievenseitnwsnctneevendsnnawdns 24 3,5, Optimization of microbial pretredtnent ccsmsccmepnasmancmmncmmancmemearmenemn 25 3.

Effect of culture medium and plH.2, Elector iqitid SOG TANG sssc-vesssenscssmsienan menmenretaseamuamreeearenesem recur 25 3. Effect of cultivation method. ‘(Constivction of complex Microbial CONSOLE ssccsseesssonnernennsasnnsnasonnrenrennenxesanevaneavounnans 26 3. Effect of quality of lignocellulosic bioimasses.

Saccharification and fermentation of pretreated biomass: cases study. 26 39, Analytical 116 thodSersccucmmnnmesusscanmupmaa are EERE 27 3.1, Détermination of deoradation tate avvccsccasnnammmnnmnnaraninemanunrmanenns 27 3. Determination of reducing SU Bai iis: cscccvenecetersurnascteeneneesonsvrsesrtannestatavin spassenieerearens 20. EnzZymalicactivity AŠŠ4WŠ‹ssoandotnnasoobDaDDOEEHCEGEDDIHESQGĐIAIGBĐSSSEENGEGINIHHMSSSSAISSEEESXSRHESS 27 3.

Determination of total phenolic €OTIf€TI{. Determination of amino acid COTIẨ€TIE. Determination of bioconversion TAÍ€. - -- -- LG 22222222 221111111 1 1 125225111111 ng xxg 29 3:10.

Statistical analysis sscsensssassensassssssnassanssnsevsnsssaves staavnssannssaseassnnsanp sae oe sensnsenamapeaaceeasssseeats 29 RESULT AND DISCUSSION ses.cnseumscrmnep arama ne aE RT 31 4. Bacterial pretreatment of wheat bran.l„ Cellulolytie Bach cree oscencanaventememmenduacmcnc mime arma: 31 41. Ligninolytic bacteria :sscssssssscsscssmsseruesssenes nares emaecmnmasmeasmeanen naa 43 4. Construction of the mixed cultures of cellulolytic and ligninolytic strains.

_ Fungal pretreatment of lignocellulosic biO1maSsS. Utilization of yeast as supplement hố. Optimization of operating parameters. Effect of culture medium and plH.

Effect of the liquid:solid raf1O. Effect of cultivation methOS. --- 6 + k1 12k TH ngàn HT TH HH 67 4. Development of the effective microbial COTSOTẨ1A.1, PrONnusiiS ap pProde hl yowcrere apie ecssiernanecrernimeennet isi aa 69 4.

Construction of complex microbial COTRSOTẨHA. Performance of newly developed microbial conSOTẨ1a. Application of newly developed microbial €OTSOTẨA. Optimization saccharification process.

Saccharification of pretreated wheat bran using mono- and co-cultures. Ethanol fermentation of biologically pretreated wheat bran. 90 Tu 1521 El IGS SNC © E2 ky sang DtoicsfifeslnrstsftrrsnssatlibreibviesloostoisosbUtonbdi noobsso=biisliiostilvoistttogdtsir 94 il Abbreviations ANOVA Analysis of Variance CFU Colony Forming Units DP Degree of depolymerization of carbohydrates FPU Filter paper unit (Cellulase enzyme activity) GDS Gram per dried substrate HPLC High-Performance Liquid Chromatography Ils Ionic liquids pretreatment OD Optical Density Organosolv pretreatment PC Principal component PCA Principal Component Analysis Refractive index SHF Separate hydrolysis and fermentation SPSS Statistical Analysis Software SSF Simultaneous saccharification and fermentation YEPD Yeast extract peptone dextrose ili List of figures Figure 2.1 Hierarchy of structures of lignocellulosic b1OfSS.-- -- 55s ss + ++x+seeesssss 4 Figure 2.2 Cellulose structure (Terzopoulou et al.3 Hemicellulose structure (Terzopoulou et al.4 Three monomer types in lignin (Duval and Lawoko, 2014).- -----+ss<csxssxsse2 6 Figure 2.6 Different lignocellulose pretreatment approaches (Abraham et al.7 Overview of biological pretreatment and its applications (Narayanaswamy et al.5 Ethanol conversion process from lignocellulosic biomass.1 Dried weight loss of wheat bran after 7-day of cultivation of Bacillus strains .2 Reducing sugar accumulation ratio of Bacillus strains after 24, 48 and 72 hours of cultivation. Capital letters (A, B, C) indicate the difference by treatment time and lower-case letters (a, b, c, d, e) demonstrate difference by SÍTA1TS.-- - - + + 2321123 9 1 1 1 1 1x ng re 32 Figure 4.3 Correlation of pH and reducing sugar yield produced in pretreatment by individual Bacillus strains B.B, coasulans BOVI39 (HĨ sncassscossaaauvenscneracmnvmennmnenemmenze meas 33 Figure 4.4 Total cellulase activity (A), endo glucanase activity (B), B-glucosidase activity (C) and xylanase activity (D) of Bacillus strains at 72 hrs of enzyme harvest .5 Cluster analysis and its characteristic using Ward’s minimum variance, based on hydrolytic enzyme and reducing sugar in pretreatment by Bacillus strains.6 Dried weight loss of wheat bran after 7-day of cultivation of Bacillus consortia .7 Reducing sugar accumulation ratio of Bacillus co-cultures after 24, 48 and 72 hours of cultivation.

Capital letters (A, B, C) indicate the difference by treatment time and lower-case letters (a; b; Gd, €) đeni6iisfFa(6:đ1ITofefice BY SA HÍẾ ocr ceccnmanenvensonaneeeeenremensacannmenmenemermnenaenonaan 40 Figure 4.8 Principal component analysis (PCA) plot (A: component plot in rotated space; B: plot of regression factor on the first and second axes from PCA of 11 Bacillus consortia) .9 Dried weight loss of wheat bran after 7-day of cultivation of ligninolytic strains .10 Comparison of enzyme production capacity of 8 lignin-degrading strains at 48 hrs of HICDĐIIGT Reeeee entre tetas ten errr ee ern rece er ert ent ere 46 Figure 4.11 The correlation between degrading enzyme activities and sugar yield.12 Total phenolic content generated by ligninolytic cOnnSOTfa.13 The sugar conversion of pretreated lignocellulosic biomass using ligninolytic strains ((A and their cosculltures: (B) ssieacocsssmexseancenantasennsauiusinesanaumsasvernaadton onasiaucasemeesnawuceltne teatien Nee 50 Figure 4.14 Cluster analysis and its characteristic using Ward’s minimum variance, based on various parameters in pretreatment by bacterial COMSOTEHIA 0.15 Enzymatic properties of three clusters I (A), cluster II (B) and cluster II (C).16 The sugar conversion of pretreated lignocellulosic biomass by bacterial consortia in three clusters (cluster I (A), cluster IT (B), cluster IIT (C)) .17 Weight loss of wheat bran by the pretreatment of fungi strains and their consortia .18 Reducing sugar vs. time of pretreatment by Ẩung1.- ---- 55 525 +22 *+2+2sc£z£zsxss 58 Figure 4.19 The sugar conversion of pretreated biomass by fungi and their consortia .20 Reducing sugar accumulation ratio of yeast strains and their consortium after 24, 48 Aid 72 HS OPCW V AOR iccsmssversnseessccareosanneermennenemenenaacen emma REED 61 Figure 4.21 The sugar conversion of pretreated lignocellulosic biomass using yeast and their co- GUNHEỂlinssekuandsissmdlsnbinanernarliseceeslidlEesaosligttdgestplielpsoiclskcbsrosiilldiposSialkaEieliusEboritialobsgsfllidigdlisitaesoollisssaiostoslEindindise 63 Figure 4.22 Degradation efficiency in biological pretreatment by fungi using different culture medium and pH values: weight loss (A), reducing sugar yield (B).- ----5+++s<+<ss++s+ss+ 64 1V Figure 4.23 Degradation efficiency by bacterial co-cultures using different culture medium (A: Weisht loss, B: reducing stpar Viel d) nrscsnscusssnasn scanner Meo 64 Figure 4.24 Comparison of enzymatic characteristics under cultivation of bacteria and fungi in submerged pretreatment (A: FPase, B:CMCase, C: xylanase and D:laccase).25 Effect of moisture in degradation efficiencies in biological pretreatment using bacteria (A) and fungi (B) oo.26 Effect of two cultivation methods on degradation efficiency in biological pretreatment of lignocellulosic biomass: Suspended (A) and submerged (B) pretreatment.27 Effect of two cultivation methods on enzymatic production in biological pretreament of lignocellulosic biomass (A suspended and B submerged pretreatment) .28 Cluster analysis of degrading criteria and their characteristics in the pretreatment by the complex consortia using Ward’s metÏod.29 The correlation between weight loss and reducing sugars from pretreated wheat ee i aca a re 74 Figure 4.30 Enzyme production by microbes at 72 hrs of pretreatimenf.31 The sugar conversion of pretreated lignocellulosic biomass.32 Weight loss of lignocelluloses using microbial consortia BFY4 (A), BFY5 (B).33 Enzyme activities after 72 hrs of lignocellulose pretreatment by microbial consortia BFY4 (A) and BFY5 0.34 Sugar conversion of different mixtures of substrate under cultivation of BFY4 (A) and BEY 5 (8) ssssenmnsacassmsancmcncnunncmmanar cm 80 9930321T83149501094498G801001143G133504E0400/5041530S549380055 80 Figure 4.35 Comparison of the reducing sugar accumulation ratio in hydrolysates after 72 hrs pretreatment then after 4 hrs hydrolysis of lignocellulosic biomass.36 Saccharification and ethanol production using mono-cultures and co-cultures (maximum 3 members) pretreated wheat bran hydrolysates. --- eeeeee tee teeteeteeenee 85 Figure 4.37 Saccharification and ethanol production using complex consortia (from 4 members and above) pretreated wheat bran hydrolysates .- -- 1 SH nh ng HH He 86 Figure 4.38 Bioconversion rate with microbial consortia using pretreated wheat bran hydrolysates eee eee re 87 List of tables Table 3.1 List of microorganisms used in this Study. ccc eceeceseceeseeeeesseceeeeeeeeseceseeeneeseeeeenees 23 Table 3.2 Description of the fungal consortia.

All synthetic microbial communities were at least 3 TC CALS cscs caeraerarrrewesereawranaie cscs as RES US WS WER Na EAD TET HETOS WN SIOE SSS HE STEN SANT SAVES ESES ISA ODEO 24 Table 3.3 Experimental design for evaluation the effect of cultivation method.4 Preparation various mixtures of lignocellulosic bioimass.1 Description of the cellulolytic COTISOTẨHA.-- 55 22+ 322332 **2E+vE+vEererrerrrrrrrrrrrrer 38 Table 4.2 Enzymatic production of various enzymes by monoculture and co-culture in the pretreatment hydrolysate at 72 hours of €uÏ(IVaf{IOTI.3 Total phenolic content (mg/ml) accumulation ratio of ligninolytic strains after 7 day WUD) oe 45 Table 4.4 Enzymatic production by monoculture and co-culture of lignin-degrading strain in the pretreatment hydrolysate at 72 hours of CuÏ{TVAfIOII.5 Degradation efficiency of co-cultures of cellulolytic and ligninolytic strains .6 Enzyme production by fungi species and their conSOTfIUI.-- -5- 55 <+ss++ss>++ 59 Table 4.7 Degradation profiles by €aSÍ. - ác t1 TH HT TH nh HH HH 62 Table 4.8 Degradation parameters (weight loss, reducing sugar accumulation, pH) of wheat bran substrate in the cultivation of mono and mixed culture in the pretreatmenf.9 Reducing sugar accumulation ratio under pretreatment of various lignocellulosic Elö†ftaS5iušlfiE Microbial CONSOTAUI res cmcamssansemerennanmamaaramarmmae aR 78 Table 4.10 Effect of substrate loadings in saccharification efficacy of pretreated biomass .11 Effect of enzyme dosages 1n saccharification efficacy of pretreated biomass.12 List of microbial consortia used in the pretreatment of lignocellulosic biomass. INTRODUCTION AND OUTLINE 1. Introduction It has been predicted that the world's population may reach 9.7 billion in 2050 and 10.9 billion in the next 50 years (Roser, 2013).

As a result of the increasing population and economic growth, by 2050, global energy consumption and energy-related carbon dioxide emissions will increase nearly 50% compared with 2020 (Energy Information Administration, 2021). Fossil fuel, natural gas, coal and nuclear energy cannot satisfy human beings' demands. The reliance on oil and gas makes the world economy dependent on the limited number of exporting countries and escalates gasoline prices. Moreover, CO2 emissions as the result of burning fossil energy from the industrial zone or means of transport vehicles have been claimed as the main reasons for serious environmental issues such as global warming, and climate change which could damage the natural ecosystem.

Thus, many governments are stimulating the utilization of renewable energies and resources to aim toward the three dimensions (three Ps) of sustainability, namely Profitability (affordable energy), Planet (climate change) and People (social stability). The renewable source will be a promising and sustainable source of energy alternatives to address the future environmental problem and energy scarcity. Different types of renewable energy are currently being extensively researched, namely solar, wind, geothermal, hydrothermal and biofuels. Among renewable energy sources, bioenergy (energy from bio-based sources) is the largest renewable energy present in the form of liquid fuels such as biofuel, diesel, or gasoline.

In 2017, bioenergy accounted for 70% of renewable energy consumption (World Bioenergy Association, 2019). Biomass can be obtained from many sources such as forestry or agriculture waste streams. There are three classifications of biofuel listed first-, second-, and third-generation biofuels.