Synthesis Of Bacterial Cellulose From Sugarcane Bagasse And Jerusalem Artichoke By Multiple Strains

Bacterial cellulose(BC)is a polysaccharide high-molecular polymer synthesized by microorganisms such as Komagataeibacter xylinus(Gluconacetobacter xylinus,Acetobacter xylinum),of which chemical essence is a cellulose molecule formed by the interconnection of?-1,4-glycosidic bond with D-glucopyranose ring as the basic structural unit.BC has excellent physical and chemical properties,such as high purity,high degree of polymerization,high crystallinity and excellent mechanical properties.In addition,BC is a kind of natural nanofiber material with high specific surface area and water holding capacity,which present hydrogel state under natural conditions.Based on its superior characteristics,BC has great application prospect in food,textile,papermaking,wastewater treatment,acoustic materials and many other fields.The high cost of BC production has always been the bottleneck of its large-scale industrial production.In the process of BC production,carbon source occupies a considerable part of the cost.The production of BC by bio-refinery using agricultural and forestry products as well as wastes as feedstocks has been expected to reduce the production cost of BC.At present,there are several main problems in the research of low-cost and large-scale production of BC.Firstly,research technology of BC-producing strains needs to be improved,and the cultivation of the strains needs to be controlled more precisely.Secondly,there are limited reports on the tolerance of strains to lignocellulose-derived inhibitors and also on the related metabolic mechanisms,which need further investigation.Thirdly,the present cultural process is relativel y conventional,and there is no research on the more simplified process such as synchronous saccharification and fermentation.Based on these aspects,sugarcane bagasse and jerusalem artichoke(JA)were used as model feedstocks to produce BC by bio-refinery using four strains of K.xylinus,DHU-HL-Z1,DHU-HL-Z2,DHU-HL-Z3 and ATCC 23770 in this thesis.The research was focused on the following three aspects:(1)Timely enumeration method and the precise control of fermentation of K.xylinus strains;(2)Research on the tolerance of different K.xylinus strains to typical lignocellulose-derived inhibitors and sugarcane bagasse hydrolysates and their convertional mechanisms;(3)Research on the preparation of BC from jerusalem artichoke tubers by acid and enzyme hydrolysates and simultaneous saccharification cultivation.Specific research contents and main results are as follows:1.Double staining method was applied on K.xylinus strains using SYBR Green I(SG)and propidium iodide(PI)to determine the viability of bacteria according to the integrity of cell membrane.The fluorescence standard curves were built for each of the four strains with a fluorescence microscope and a fluorescent plate reader.The R~2 values of the four strains range from 0.9707 to 0.9757,which indicate good linear correlation of eight independent experiments.The timely enumeration method was used to precisely control the initial cell concentration of fermentation,so that the initial viable cell concentration of four strains could be accurately controlled at 5×10~6 cell/mL,and there was no significant difference in the initial cell concentration(p>0.05).On the premise of precise control of cell numbers in the inocula of four strains,the differences of BC yield and properties were compared.BC yields of DHU-HL-Z1,DHU-HL-Z2 and DHU-HL-Z3 were 12.82±0.18 g/L,12.93±0.49 g/L and 14.78±0.43 g/L,respectively.There was no significant difference among them(p>0.05).The BC yield of K.xylinus ATCC 23770 was8.65±0.83 g/L,which was significantly lower than that of the other three strains(p<0.01).BC produced by the four strains showed infrared characteristic peaks of cellulose in FT-IR spectra.Among them,the BC from DHU-HL-Z2 and DHU-HL-Z3 had better mechanical properties and higher Young modula,which were 0.130 Mpa and 0.096 Mpa,respectively.The crystallinities of the BC from DHU-HL-Z1 and DHU-HL-Z2 were 87.64%and 86.65%,respectively.The average fiber diameter of the four strains was 37-48 nm.2.The tolerance of four strains DHU-HL-Z1,DHU-HL-Z2,DHU-HL-Z3 and ATCC 23770to lignocellulose hydrolysate was investigated by using four model inhibitors,furfural,5-hydroxymethyl furfural,coniferyl aldehyde and vanillin.DHU-HL-Z3 and ATCC 23770 were less affected by 10 mmol/L furfural,and BC production was 56-60%of the corresponding control group without inhibitor.The BC yield of DHU-HL-Z1,DHU-HL-Z2 and DHU-HL-Z3 in the experimental group containing 15 mmol/L 5-hydroxymethyl furfural(HMF)decreased to 80%of the corresponding control group,but the absolute yields of the three strains were still higher than that of ATCC 23770.One mmol/L of coniferyl aldehyde only prolonged the lag phase in growth of four strains,and had no significant effect on BC production.In the culture medium added 2mmol/L vanillin,the cellulose yields of DHU-HL-Z 2 and DHU-HL-Z3 were more than 90%in the corresponding control group,and the tolerance of which was better than t hat of the other two strains.The inhibitors were transformed by four strains.The main bioconversion products of the four strains were the same,but there were some differences in the conversion rate.Furfural was oxidized to furoic acid,and the bioconversion yields of the four strains ranged from 38%to 78%.5-hydroxymethyl-furfural was oxidized to 5-hydroxymethyl-2-furoic acid,and the bioconversion yields of the four strains ranged from 49%to 100%.coniferyl aldehyde was oxidized to ferulic acid,and the bioconversion yields of the four strains ranged from 31%to 38%.The bioconversion oxidation and reduction products of vanillin were coexisted,which were vanillic acid and vanillic alcohol,respectively.Vanillic alcohol was the main conversion product.The conversion rates of the four strains were very different.The conversion rate of DHU-HL-Z 3 was 80%,and the other three strains were between 13%and 28%.The secondary biotransformed product of vanillin was vanillic acid,and the conversion rates of the four strains were between 10%and 14%.There was a positive correlation between bacterial strain bioconversion to inhibitor and its tolerance to inhibitor.The bioconversion products were less toxic than the original substances,which could be regarded as the process of biological detoxification.The difference in tolerance of the four strains might be related to the expression of oxidoreductases and related resistance genes in their cytomembrane.Taking sugarcane bagasse hydrolysate as a complex inhibitor model,the tolerance differences of the four strains were further studied.The sugarcane bagasse hydrolysate contained 96.1 mmol/L furfural and 8.7 mmol/L 5-hydroxymethyl furfural.Experimental results showed that the strains could not tolerate more than 15(v/v)%sugarcane bagasse hydrolysate.15(v/v)%sugarcane bagasse hydrolysate delayed the sugar consumption of the four strains,and 10(v/v)%and 15(v/v)%of the sugarcane bagasse hydrolysate decreased the living cell concentration of the four strains.Sugarcane bagasse hydrolysates of 10(v/v)%and 15(v/v)%resulted in BC yields with improvements from 21.1%to 39.1%for strains DHU-HL-Z1,DHU-HL-Z2 and DHU-HL-Z3,and almost no yield improvement for strain ATCC 23770.3.With jerusalem artichoke tubers as feedstock,bacterial cellulose was produced by using hydrolysis followed by cultivation.The crude BC yield from the culture medium containing JA-acid hydrolysate was 4.7±0.1 g/L,which was significantly higher than 2.1±0.4 g/L of fructose control group(P<0.05).The crude yield of BC was 12.1±1.1 g/L in culture medium containing JA-enzyme hydrolysate,which was significantly higher than that in the other two experimental groups(p<0.05).In terms of mechanical properties,the Young modulus of JA-enzyme group was0.84±0.05 Gpa,which was significantly higher than 0.61±0.13 Gpa of JA-acid group(p<0.05).The JA tubers were used as a raw material to establish a simultaneous saccharification and fermentation(SSF)process.Firstly,the optimal process conditions for SSF are as follows:temperature 30°C,the initial pH 5.0,JA dry mass concentration 50 g/L,and 1300 U/L inulase.Two strains of DHU-HL-Z3 and ATCC 23770 were applied to evaluate BC production process via SSF.The results showed that pure BC yield of strain ATCC 23770 in SSF group was 3.14±0.06g/L,which was significantly higher than(p<0.05)fructose control group,1.17±0.04 g/L.The pure BC yield of strain DHU-HL-Z3 in the SSF group was 7.16±0.29 g/L,which was significantly higher than that of fructose control group(3.87±0.11 g/L).Compared with the control group,the mechanical properties and crystallinity of the SSF group had significant improvements.In summary,the innovation points of this thesis mainly include the following aspects:(1)Timely enumeration method for BC-producing strains has been established,and applied to the precise control of living cell concentration at the beginning of cultivation of four K.xylinus strains.(2)With the precise control method,differences in tolerance of the four strains to typical lignocellulose-derived inhibitors(namely furfural,hydroxymethyl furfural,coniferyl aldehyde and vanillin)were studied for the first time,and the bioconversion of the inhibitors has been investigated.(3)Simultaneous saccharification and fermentation process has been realized in BC production using jerusalem artichoke tubers as a model feedstock,and the yield and properties of BC have been significantly increased with the process.The study addressed critical problems in the field of bio-refinery of bacterial cellulose and will lay a foundation for subsequent researches.