| Fumaric acid is found as the simplest unsaturated binary carboxylic acid so far.Due to the unsaturated double bond and two carboxylic groups in its structure,fumaric acid could be further derivatized and functioned,which means that it has great application potential.At present,the production of industrial fumaric acid mainly relies on chemical methods.However,with the increase of oil price and the gradual emergence of environmental problems,the disadvantages of chemical methods are increasingly prominent.The microbial transformation to produce fumaric acid from renewable biomass has attracted much attention due to its mild conditions,environment-friendly and small variety of by-products,which has become the most promising method for fumaric acid production.Lignocellulose was the most abundant renewable resources in nature,but there was almost no research about organic acid production using it as raw material in the fermentation process.In this research,we started from solving the problem of efficient utilization of xylose through strain screening from Rhizopus arrhizus RH 7-13 saved in our laboratory.And then the strain was used in the fermentation of lignocellulose hydrolysate to develop a microbial method to produce fumaric acid and enrich the variety of substrates for microbial transformation at the same time.In addition,according to the fermentation process to produce high value biological chemicals by microorganism,they were found strictly affected by the carbon/nitrogen ratio of the medium.Therefore,Yarrowia lipolytica,due to its clear genetic background and numerous available operation tools,was chosen to explore transcription of the key genes in metabolic pathways and to explain the process of gene expression regulated by carbon/nitrogen ratio.The detailed research content of my research as followed:1.Rhizopus strains with high utilization of xylose were screened and the co-fermentation process of xylose and glucose was studied.Fumaric acid production ability of Rhizopus arrhizus RH 7-13 using xylose was firstly investigated and the titer of fumaric acid was low.The medium with high concentration of xylose was used to do strain screening.The new strain Rhizopus arrhizus RH 7-13-9#was obtained and 45.31 g/L fumaric acid was produced in 80 g/L xylose fermentation by immobilized mycelium on carrier,which was 1.7 times higher than the production from original strain Rhizopus arrhizus RH 7-13.The conversion rate of xylose reached 73%,which was the highest level reported so far.To investigated fumaric acid production ability of the new strain from glucose,80 g/L glucose was used as solo carbon source in the medium and fumaric acid yield can reached 37.52 g/L,which meant that the new strain could efficiently utilize both glucose and xylose.On the basis of it,co-fermentaion of xylose and glucose process with tatal carbon source 80 g/L was conducted.In this process,it was found that when the glucose/xylose ratio was 75%:25%and carbon/nitrogen ratio was 800:1,we could get the highest fumaric acid production 46.78 g/L.Compared with the result of glucsoe as solo carbon source,it was increase by 25%,which laid a good foundation for the utilization of lignocellulose hydrolysates.2.The production of fumaric acid from lignocellulosic hydrolysates fermentation by Rhizopus arrhizus RH 7-13 was studied.The Dried Distiller’s Grains with Solubles as by-product in ethanol production was used as feed-stock to prepare lignocellulose hydrolysates by traditional acid hydrolysis method.The pretreated detoxified lignocellulose hydrolysate was used carbon source to produce fumaric acid by Rhizopus arrhizus RH 7-13-9#.It could grow normally in the pretreated detoxified lignocellulose hydrolysate and 21.03 g/L fumaric acid was produced by pellet morphology using concentrated detoxified lignocellulose hydrolysate.After adding 47.13 g/L glucose and 13.75 g/L xylose,the ratio of glucose/xylose in the hydrolysate reached 75%/25%and the fumaric acid production reached 30.13 g/L by immobilized mycelium on the carrier,which was close to titer(37.52 g/L)using 80 g/L glucose as solo carbon source.Unpretreated lignocellulose hydrolysate was also try to be applied into fumaric acid fermentation process,which was adjusted by KOH to pH 4.7,and could be directly used as seed culture medium for Rhizopus arrhizus RH 7-13-9#.It was difficult to use to produce fumaric acid but benefit to produce ethanol.When 60 g/L glucose was mixed with unpretreated lignocellulose hydrolysate used as medium,ethanol production reached 29.33 g/L with a space-time yield of 2.44 g/L h.When the concentrated unpretreated lignocellulose hydrolysate used as medium,ethanol yield reached 16.26 g/L with a space-time yield of 1.36 g/L h,which was higher than the result of Saccharomyces cerevisiae fermentation of lignocellulose hydrolysate.3.The production process of fumaric acid from Rhizopus arrhizus RH 7-13-9#in stirred-tank reactor was optimized.When fumaric acid was produced using pellets morphology in 5 L stirred-tank bioreactor,the pellet morphology was difficult to control in the stirred-tank fermenter and part of the pellets were scattered,winding on the impeller,blocking the vent and sampling connection at the end of fermentation process.In order to solve these problems,we tried to conduct mycelium immobilization fermentation process in stirred-tank fermenter.Comparing mycelium immobilized on different carriers,it showed that mesh biomass materials(loofah fiber)was the most suitable carrier.After fermentation process optimization,it indicated that when the level of carrier was 6.5 g/L and stirring speed was 400 rpm,the fumaric acid yield reached the highest 36.69 g/L with a space-time yield 0.437 g/L h.At the same time,all the mycelium could completely attach to the carrier,which avoided the control of the pellet morphology,and solved the problems such as the intolerance to shear force,pellet easy to break up,and mycelium easy to block the vent.On this basis,the continuous batch fermentation process was realized in the stirred tank fermenter in this research.The total concentration of fumaric acid was 25.2 g/L in three batches fermentation process,and the fermentation time was shortened by about 50 h compared with the single batch fermentation process.Detoxified lignocellulose hydrolysates instead of part of carbon sources was also used to produce fumaric acid by this method in the stirred-tank fermenter,and 25.03 g/L fumaric acid was obtained,which further proved that lignocellulose hydrolysates could replace part of carbon sources in the fermentation process for the production of organic acids.4.The effect of carbon/nitrogen ratio on gene expression was investigated using auxotrophic Yarrowia lipolytica Polg(Leu-)as original strain.PYLXP ’-Nluc expression plasmid was constructed by cloning the promoter of 22 key genes in the metabolic pathway of Yarrowia lipolytica strain with luciferase Nluc as the reporter gene.The expression plasmid PYLXP’-Nluc was transferred into Yarrowia lipolytica Polg(Leu-)and cultured in the medium with the carbon/nitrogen ratio of 10:1,50:1 and 100:1 to detect luciferase activity.22 key genes was divided into four modules according to their function and metabolic pathway to analyze promoter activity,namely NADPH regulation module,the carbon skeleton regulation module,lipid synthesis and degradation modules and TCA-glyoxylate cycle regulation module.The activity of 15 genes promoters were found improved with the increase of carbon/nitrogen ratio,including ZWF1,GND2,MnDH1,MnDH2,MAE1,UGA2,PDC1,HXK1,FAS2,DGA1,POX4,IDH2,ICL1,UGA1,GAD,which meant that the transcription of these key genes were affected by the carbon/nitrogen ratio.These results could explain the reason why the strain showed different fermentative activity in the accumulation of lipids under different carbon/nitrogen ratio.And it laid a foundation for further study on the effect of carbon/nitrogen ratio on the metabolic process of Rhizopus arrhizus. |
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