Study On Production Of Bacterial Cellulose From Winey Pomace Fermented By Komagataeibacter Rhaeticus K15 And Its Reductive Adsorption Of Chromium

Bacterial cellulose（BC）which is produced by bacteria has the same chemical structure with plant-derived cellulose.Due to its unique properties such as high purity,high crystallinity,modification accessibility and good biocompatibility,BC has attracted more attention and is widely used in food,cosmetics,and materials research.However,the industrial application of BC is now limited by the production capacity of strains and the cost of culture medium.Screening strains with high productivity and exploring low-cost medium are the critical solutions to solve these problems.The bioconversion of agricultural waste to BC is a widely studied strategy to effectively reduce the production cost of BC.Nowadays,the scale of wine industry in China is expanding rapidly,while a large amount of winey pomace is produced accompanied with the production of wine.The aim of this research is to screen a productive cellulose-producing strains and use the winey pomace as a low-cost medium to promote the bio-conversion of into BC.The overall review of this research is listed as follows:1.Isolation and identification of the productive cellulose-producing strains from KombuchaBased on the similar fermentation environment between winey pomace and Kombucha（both rich in polyphenols）,four cellulose-producing strains were isolated from Kombucha,and identified by morphology,physiological and biochemical tests and molecular biological identification method.In addition,the fermentation characteristics of all four strains were studied,as well as the characterizations of fermented products（BC）.Four strains were screened from Kombucha,named as Komagataeibacter oboediens K2,Novacetimonas hansenii N7,K.xylinus K13,and K.rhaeticus K15.Fermentative profiles results showed that medium p H dropped to about 3.5 during the initial fermention stage of K.oboediens K2,N.hansenii N7,and K.xylinus K13.However,we found the medium p H during the fermentation of K.Rhaeticus K15 maintained a stable value（about 5.0）,and thus speculated that this stable p H might be related to the production capacity of gluconic acid in K15 strain.In addition,SEM,FTIR,and XRD results indicated that the fermentated products of four strains had typical characteristics of cellulose,while BC synthesized by N.hansenii N7 and K.xylinus K13 had lower crystallinities as demonstrated by the multi-crystalline peaks.Based on the above results,K.Rhaeticus K15 was selected as the target strain for the next study.2.Metabolic pathway analysis and functionality validation of of K.rhaeticus K15 based on genome-wide sequencingThe metabolic pathways and cellulose synthesis operon of K.rhaeticus K15 were analyzed by genome sequencing.Sequencing results showed that the genome was a total of310 contigs contributing to the length of 4,028,747 bp long with the GC content of 62.78%.There were 3791 coding DNA sequences（CDS）were predicted（average length:907.81 bp）.A total of 43 t RNA and 3 r RNA were predicted in the K15 genome.A total of 5 operons were annotated in K15.Based on KEGG prediction of BC anabolic pathway,it was confirmed that the gene encoding the cofactor pyrroroquinoline quinone（PQQ）in K15 was incomplete,leading to the inability to synthesize glucose dehydrogenase.This result explains why K15produces less gluconic acid and higher BC yield during fermentation.Carbon source experiments proved that the K15 strain produced little gluconic acid（2.26±0.12 g/L）and higher cellulose production（4.76±0.17 g/L）after 10 d of fermentation.Furthermore,six nitrogen-fixing genes were found to be responsible for the survival of K15 on a nitrogen-free medium.The BC yields were higher when glucose,fructose,sucrose,mannitol and glycerol was used as the sole carbon source compared to lactose.Besides,the SEM,XRD,and FTIR results indicated that the carbon source was of little significance in affecting the BC structure.3.Preparation and characterization of BC from winey pomaceCellulase and pectinase were used to hydrolyze winey pomace,respectively,and the optimal enzymatic hydrolysis conditions were obtained by 3×3 factor Box-Behnken design experiment and response surface analysis.After component analysis,the enzymolysis solution and impregnation solution were fermented with K15 with HS medium as control.The results verified that the reducing sugar content of enzymatic hydrolysis solution was19.36±0.36 g/L.The content of reducing sugar,glucose,sucrose and total phenol in the enzymolysis solution was significantly higher than those in the impregnation solution.Amino acid composition results revealed that there was no significant difference in amino acid content between enzymolysis solution and impregnation solution.The results of phenolic composition showed that monomers with high water solubility such as catechin,epicatechin and gallic acid had higher proportions both in the fermentation liquid and enzymatic hydrolysate.We found that in the early stage of fermentation,the metabolism of K15 in the hydrolysate was slow due to the antibacterial effect of polyphenols.However,after adapting to the environment,K15 strain had a similar fermentation profiles with control medium,and the yield of BC was 4.28±0.21 g/L（dry basis）after 10 d of fermentation.The structure and functional properties of BC synthesized from winey pomace were characterized by SEM,FTIR,XRD,DSC,and antioxidant activities of these cellulose were also evaluated.The cellulose concentration of BC produced by winey pomace was higher than 90%,and meanwhile had characteristic peaks related to polyphenols as indicated by FTIR,which proved that grape polyphenols was in-situ combined with BC.XRD results showed that the crystalline pattern of BC from winey pomace was the same with that produced by HS medium,while the crystallinity of BC produced by winey pomace was higher.In addition,BC produced by winey pomace had wider fiber diameter distribution.The thermal stability of BC produced by winey pomace was lower than that produced by HS medium,as proved by the endothermic and exothermic peaks related to lignin at 150-200℃in DSC curves.Most importantly,BC produced from winey pomace could load more free phenols compared to bound phenols,and had a slower release of polyphenolst and higher antioxidant activity.4.Adsorption performance of BC from winey pomace on hexavalent chromiumThe adsorption performance of BC gel synthesized from the enzymatic hydrolysate of winey pomace on Cr（Ⅵ）in water was studied,and the mechanism of adsorption for Cr（Ⅵ）was revealed by FTIR,XRD,XPS,and SEM-EDX analysis.The adsorption efficiency of BC on Cr（Ⅵ）in water increased with the decreased p H.The optimal adsorption effect was achieved when p H=1,and the maximum equilibrium adsorption was 473.09±1.41 mg/g（db）.Coexisting cations had little effect on adsorption performance,while the addition of NO₃^-and SO₄^2-significantly reduced the equilibrium adsorption capacity of BC gel for Cr（Ⅵ）.FTIR spectra showed that the adsorption did not alter the structure of cellulose,while absorption peaks related to Cr（III）and Cr（Ⅵ）appeared after the adsorption of Cr（IV）by BC.XRD showed that Cr（Ⅵ）did not change the crystalline pattern of BC,but a new crystallization peak assigned to Cr₂O₃ appeared in BC after the adsorption of Cr（Ⅵ）.According to XPS analysis,the adsorbed BC contained both Cr（Ⅵ）and Cr（III）.SEM-EDX analysis showed that multiple Cr peaks with different intensities appeared in the EDX spectrum of adsorbed BC.Combined with element content analysis,the content of C decreased significantly after adsorption,while the content of element O increased slightly.This study not only provides a new idea for BC production of agricultural waste,but also broadens the application range of BC.