MINISTRY OF EDUCATION AND TRAINING NONG LAM UNIVERSITY HO CHI MINH CITY FACULTY OF BIOLOGICAL SCIENCES PRODUCTION OF BACTERIAL CELLULOSE BY Komagataeibacter xylinus USING AGRICULTURAL WASTES IMPREGNATED WITH SILVER NANOPARTICLES GREEN SYNTHESIZED FROM Eclipta prostrata EXTRACT Major: BIOTECHNOLOGY Student: LE THI MY HOA ID: 17126197 Academic year: 2017 — 2021 Thu Duc city, 03/2023 MINISTRY OF EDUCATION AND TRAINING NONG LAM UNIVERSITY HO CHI MINH CITY FACULTY OF BIOLOGICAL SCIENCES UNDERGRADUATE THESIS PRODUCTION OF BACTERIAL CELLULOSE BY Komagataeibacter xylinus USING AGRICULTURAL WASTES IMPREGNATED WITH SILVER NANOPARTICLES GREEN SYNTHESIZED FROM Eclipta prostrata EXTRACT Supervisors Student Dr. BIEN THI LAN THANH LE THI MY HOA Dr. NGUYEN THI THANH THUY Thu Duc City, 03/2023 ACKNOWLEDGEMENT First and foremost, I would like to express my sincere gratitude to my research supervisors, Dr. Bien Thi Lan Thanh and Dr.
Nguyen Thi Thanh Thuy for giving me invaluable advice, strong support and patience during my undergraduate thesis time. Besides, I would also like to thank B. Nguyen Quoc Thang for sharing his statistics knowledge and giving me helpful advice. Without their assistance and dedicated involvement during the process, my undergraduate thesis could have never been accomplished.
I would also like to acknowledge all lecturers at the Faculty of Biological Sciences, Nong Lam University Ho Chi Minh City, have enthusiastically communicated to me knowledge during the four years of teaching and training. Moreover, I would like to thank all the members of the Laboratory of Microbiology - Room Bio 311, who always supported and encouraged me whenI had trouble. Last but not least, my deepest gratitude would go to my family, especially my parents, who not only extended their assistance to me to finish my undergraduate thesis but also gave relentlessly emotional support. AFFIRMATION AND COMMITMENT My name is Le Thi My Hoa, student ID: 17126197, class: DH17SHC (Phone: 0363123420, Email: 17126197@st.vn), of Biotechnology, Nong Lam University Ho Chi Minh city.
This is my undergraduate thesis which was conducted by myself. All the data and information are completely honest and objective. I take full responsibility in front of the committee for these commitments. Thu Duc City, February 6", 2023 Student’s signature Le Thi My Hoa ABSTRACT Bacterial cellulose (BC) has attracted more attentions as a biomedical material due to its unique properties.
However, the BC production is a relatively expensive process and BC itself has no antibacterial activity. This study aimed to produce BC by Komagataeibacter xylinus using agricultural wastes, and then impregnated with green synthesized silver nanoparticles (AgNPs) for further application in wound dressing production. Among three agricultural wastes including jackfruit rag, watermelon rind and dragon fruit peel, jackfruit rag is the most suitable substrate chosen for optimizing culture conditions. Response surface methodology with orthogonal central composite design was used to optimize Brix, pH and temperature.
The optimal conditions were found at 34°C, Brix 8 and pH 5.5 resulted in the highest dry weight of BC of 2. Biological activities of AgNPs, including antibacterial activity, minimum inhibitory concentration (MIC) and antioxidant activity were determined. The AgNPs showed antibacterial activity against both tested Gram - positive and Gram - negative pathogenic bacteria. MIC value of AgNPs against S.
aeruginosa ATCC 9027 and E. coli ATCC 25922 was 0. Their antioxidant capacity was five times less than ascorbic acid. To possess antibacterial activity, BC was soaked into AgNPs solution via ex s7 method to produce BC - AgNPs composite.
The highest antibacterial activities of the composite were observed at an initial AgNPs concentration of 120 ppm. Key words: agricultural waste, bacterial cellulose, AgNPs, RSM, antibacterial activities CONTENTS Page JNGKNOWLEDGBNHENL6siecsáes516116522664006010101633801G0E39E3013EIGSS3SAlEJ3SB4ã8S3SZ3084B43E34g380:30g0ig3-5 1 AFFIRMATION AND COMMITMENT ssssssssssssssnsseserscancsevaen case ccnevesssontsaasnssaseesevesneess il RS ERC tt ee Ee it SO eS SSD rh SH SRSA 1H TABLE OF 60) 61010010157Ẻ7. 1V LIST OF ABBREVIATIONS. S22 2H HH HH HH tre vu LIST OF TABLES ncccunnaenunemw acre ne te BEE EES Vili LIST OF BIGURES neeannnndrinbtoeosssitoSREDDHSSGIESSIGEGSREEGISEDEEHSGSSE-SESSEGNHGIGEEESEDGB8G8530083 0H03 1X CHAPTER.
Contents c2ixzt8t101101218001029/8055/00880L537XENSaHBELENSia2,053S8A4830)/4g03880gIA3. Introduction of Komagataeibacter XWÏÏHS.LHSHOEV Bild TAR ODOM Y son bá G13 nhà hột 48156 5610 cóc snes S85:GE330588gi38100183036035)-4ã60336861:0835. 3 2 Add C HĐYRGIELISHSicossysssositiSxsSN0SILSGHLEBGBEESIIGIJESBIGHEESLSESIBESEBSGSISHEIGRRGEAENGSE2J834835.300388 3 2:2, Bacterial cellulose:o Vervile Wiss sccsessx-2045656623038644380936458885 6 1588838s4586401181385084583888,60358364) 4 2. Cellulose producing baCf€T1a.-- i62 S132 2 E2 xxx HH re 4 2.
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Properties and Characteristics .-- 5 - + + 2+ 2x 2xx ngư 10 2:3:3: Mechanism of antimicrobial HOT sxsssssssssssizsssesstz5153139815580035559361001850405585983 10 2. Synthesis of AgNPs by plant €XÍTAC(.- - -- c2 2x S12 1S HH HH re 11 2/4. In situ and ex situ method for production of BC - AgNPs composife. Properties and applications x cavessccs vases sonepscavena aces esuenaneuerenene en EEE 12 2.
Response surface methodology (RSM) wescvessassecssuvereqeeseeneseeemecverreawenieeenveees 13 DSicers ALLE OC ULC Ol cecsincronastnanrmenssoaeanasivne cre ion ero i Steams ONT 13 2. Central composite design (CCD)). Recent researches in Viet Nam and the world .1, Recent researches in the world wuscancsss rarer nee 14 2.022 RESSATCHES Afi VIGENNHEssseeeeseeeiosietiidotbtrioDBISSDGLSTSBEE01SGGD.RUIESBEGDISIESE2DSHGEE 14 CHAPTER 3. MATERIALS AND METHODS .cccccececeeseeeeseeseececeeeeeeeeeeeeaeenees 16 Sele LUGS Aid PIACE OL SHIA Y sssccrenanucnseeaerin aren mTOR RRA ERR 16 3.
Materials atid THEUHGđRS. <6 S222 2 002 1 n2 Hà 4 g2 CÓ HH GÌ g3 HH 00823010888. cg0e 16 Bide lhe: NV TBÍCT- NI s:22125:550 050602 88161L/D8ÓGEHSSBES.GIGEESSSSSEENHSAG/SISNGI38/ĐEENNSGESSEENGESSGS8H088-10588 16 3. Evaluating the effect of agricultural wastes on bacterial cellulose (BC) DP OCUIC (OT ec cxcosszensseeneneornasennessx sameeren sansa aces nurse ameuesuina ime aaneseUReeE RRR ER TERRIER 16 3.
Evaluating the effect ofbacterial culture conditions on the BC production RVC s5 sg80065 opine eamesmeiere cra eer oe ret ra crn ra 17 3. Biological activities of silver nanoparticles (AgNPs) green synthesized using J52101271277251127121>sie PS San.1 ADUDAC(EEIA] ACV IY oc sncnncorecscw emcee thùhGilitatogshSSiENGt0g133:000030919018.8S30HNiĐQSEGTSEEĐSIN088 19 3. Determination of minimum inhibitory concentration (MIC).- 19 Seeded Sil DP PEL ASS AY secsesas61458005S232096:388:0559048093E0'0EGS8S0BE.SSEBGGISH/GU20800131ĐSEGĐS5GGI23. Evaluating the antibacterial activity of AgNPs impregnated bacterial cellulose 1210116) 621 (ae eee ee ee nee ee ee eee 20 32/5, SUAUSIC al AIA VSO ies cccr cece serene tye eerie oot ar cee neat aseres cos aun guieent generational 2) CHAPTER 4.
RESULTS AND DISCUSSIONS. The effect of agricultural wastes on bacterial cellulose (BC) production. The effect of bacterial culture conditions on the bacterial cellulose production ple) ee 23 4. Biological activities of silver nanoparticles (AgNPs) synthesized by Eclipta PV OSTPOTACRITACE.
«sncienanesennantann tannstesnnstueshansttiinsh anbsbnnn nnnsbinansannbinaahasnivannasiiineancseeandinesonnensrss 28 4. Minimum inhibitory concentration (MIC) of AgNPs. DPPH ASSAY seccsscceswsmseceve evassueevessensavansnavcveseavnsunncunvewaupanneveneaexansasevaeneouwsveseswanwess 31 4. The antibacterial activity of AgNPs impregnated BC membrane.
CONCLUSIONS AND SUGGESTIONS. SUB PeSTONS ceuncn uw eueesmee ee RE EE EEE Ee REEO REFERENCES eeereireebissiieniEEEEEG04031194156090123536159108565359146410011E41014316039014088 39 APPENDIX vi LIST OF ABBREVIATIONS AAB :Acetic acid bacteria AgNPs :Silver nanoparticles ANOVA :Analysis of Variance ATCC :American Type Culture Collection BBD :Box - Behnken design BC ‘Bacterial cellulose BC - AgNPs :Silver nanoparticles impregnated bacterial cellulose BNC :bacterial nanocellulose CCD :Central composite design CLSI :Clinical and Laboratory Standards Institute DNA :Deoxyribonucleic acid DPPH :2,2-dipheny]-1-picryl-hydrazyl-hydrate DRF :Dragon fruit peel FDA :Food and Drug Administration HS :Hestrm - Schramm -Jackfuit rag MHA :Mueller Hinton agar MHB :Mueller Hinton broth MIC :Minimum inhibitory concentration p-INT :p - 1odonitrotetrazolium violet solution ppm :Part per million RSM :Response surface methodology SEM :Scanning Electron Microscope UV-Vis :Ultraviolet - Visible WR :Watermelon rind ZOI :Zone of inhibition vii LIST OF TABLES Page Table 3. The coded and actual values of experimental variables used in the central composite (CCD - Orthogonality) design .(6ST071/17i8DE cesarean aati es a csc eC 17 Table 4. The actual results of reponse value (dry weight) from central composite design matrix based €X€TIIT€TIS.
The IC50 value of AgNPs, Eclipta prostrata extract and ascorbic acid. Mean ZOI of BC - AgNPs against the four pathogenic bacterial strains .36 viii LIST OF FIGURES Page Figure 2. Chemical structure of microbial cellullose (Lustri et al. The dry weight of BC from the three waste SOUTC€S.
Dry membranes of bacterial cellulose at constant weight. Correlation of the dry weight between the actual and predicted value. The ANOVA for quadratic model.-- -----+++s+<+s++se<sxsseszeeses 26 FEieure 4:5: Fit statistics of the model s:ccscscmsupesssemu mann aa eR 26 Figure 4. The optimal condition with desirability of 0.952 for the BC production.
SEM micrograph of bacterial cellulose produced at the optimal condition. Antibacterial activity assay against E. aureus ATCC 25923 and B. Microdilution method for MIC determination of AgNPs before and after 8EDIVIHTGTP-TLE Ự EbooistesesssiatfroiGidoiolliEsaSlggiE-GGHGEESIEHEEUSS/EUEBSEB28:483894082853080u81 2300810953380 31 Figure 4.
The DPPH scavenging activity of AgNPs (A), Eclipta prostrata extract (5986. The produced BC-AgNPs composIfe. The SEM image of the BC-AgNPs composIfe. Antibacterial activities of BC-AgNPs at different concentration of AgNPs against S.
aeruginosa ATCC 9027 (C) and E.ecececcesceseesseceeceeesceseeseesceseescsceeceeceecseceeseesesaeseesaseeeeseeseeees DD Figure 4. ZOI of BC-AgNPs at different concentration of AgNPs against S. aeruginosa ATCC 9027 and E. Problem statement Bacterial cellulose (BC) is a type of natural cellulose synthesized by microorganism.
It possesses some distinguished physical properties such as high porosity, excellent water holding capacity and good biocompatibility, making it widely used as a building block for biomedical applications such as wound dressings and artificial skins, artificial blood vessels and biomembranes. BC is possibly a good candidate for wound dressing as well, because it provides moist environment in wound healing. However, BC itself does not have any antibacterial activity to prevent wound infection, hence, it needs to supplemented with antibacterial agents such as antibiotic, AgNPs, etc. The level of resistance, especially against antibiotics is still increasing.
Thus, new approaches to overcome bacterial resistance are being tried. AgNPs have typical physical and chemical properties as high thermal and electrical conductivity, surface - enhanced Raman scattering, etc. These properties take AgNPs to the top of the priority list. Green synthesis of AgNPs by plant extract is a safe, clean and rapid technique (Elhawary et al., 2022; Krishnan et al.
Moreover, BC production is a relatively expensive process, due to the use of expensive culture media. The culture medium represents approximately 30% of the total cost. Therefore, one important aspect of the fermentation process is finding out a new cost - effective culture medium that can give high yield. According to Minister of Agriculture and Rural Development, Viet Nam wastes 160 tons of agricultural by - products a year.
Utilizing agricultural wastes as carbon source for the BC production is potential and eco - friendly approach. However, data about this approach is still limit in Viet Nam. Therefore, this study was conducted to produce the BC from agricultural wastes and then impregnated with AgNPs synthesized by plant extract to produce BC - AgNPs composite as an antibacterial material for further application in wound dressing production. Objectives This study aimed to produce the BC from agricultural wastes and then impregnated with AgNPs to produce nano composite for further application in wound dressing production.
Evaluating the effect of agricultural wastes on the BC production. Evaluating the effect of bacterial culture condition on the BC production. Biological activities of AgNPs synthesized by Eclipta prostrata extract. Evaluating the antibacterial activities of AgNPs impregnated BC membrane.
Introduction of Komagataeibacter xylinus 2. History and taxonomy Formerly, bacterial cellulose (BC) was described by Brown in 1886, when he found a jelly - like translucent mass on the surface of vinegar fermentation broth produced by the strain called as Bacterium aceti (Brown, 1886). It has since been known as some other names, mainly Acetobacter xylinum and Glucoacaetobacter xylinus (G. xylinus) (Dourado et al.
xylinus was reclassified as Komagataeibacter xylinus (K. xylinus) according to its 16S rRNA gene sequences. Komagataeibacter was named in honnor of Dr. Kazuo Kamagata (The University of Tokyo, Japan), who contributed to the bacterial systematics, especially of acetic acid bacteria (Yamada et al.
The word “xylinus” derived from xylon Greek nom., etymologically means woody. xylinus belonged to acetic - acid producing genera and the family of Acetobacteraceae.