Itaconic Acid Production From Forest Residues By Aspergillus Terreus

Itaconic acid(IA)is recognized by the US Department of Energy as one of 12 high value-added chemical platform compounds derived from biomass.It is widely used in many fields,such as water treatment,adhesives,synthetic resins and pharmaceuticals.The main production method of itaconic acid is bio-fermentation with the raw material of mainly sugar-based and starch-based carbohydrates,which brings high cost.At present,the efficient use of agricultural and forestry residues has become a hot spot.Forest residue resources have the advantages of vast sources,cleanliness and sustainability.However,these residues have long been discarded,incinerated or landfilled,which not only wastes resources but also causes environmental pollution.So far there have been few reports on the production of itaconic acid by the fermentation of forestry residues.Taking into account the shortage of food resources in our country,the research on fermenting acid production with forestry residues has urgent significance.The use of forestry residues for the itaconic acid production needs to solve the problems of degradation-resistant lignocellulosic structures and low acid productivity of strains,etc.The objective of the study is to obtain itaconic acid from forestry residues using microbial fermentation technology.The recalcitrance to enzymatic hydrolysis of the lignocellulose was reduced and the hydrolysis efficiency was improved by physical and chemical combination of pretreatment process.Mutation breeding was conducted with composite mutagenesis techniques to obatin strain with high yield of itaconic acid.And a complete high-density fermentation process was obtained through process optimization and metabolic engineering.The efficient utilization of forestry residues for preparing itaconic acid was achieved.The study provides a reliable data foundation and technical support for the industrial production of itaconic acid from forestry residues.The specific findings are as follows:(1)Breeding of strain with high-yield of itaconic acid by compound mutation coupling high throughput screening: High-throughput screening with 48-well microplate of Aspergillus terreus was established and the mutagenesis selection of Aspergillus terreus CICC2433 was performed using a compound mutagenesis technique.First,a mutant strain AtUV3-325 was obtained by UV-LiCl mutagenesis with the itaconic acid production of 30.02 g/L,which was 59.43% higher than the original strain of 18.83 g/L.Subsequently,a strain of AtDES3-235 was obtained through further mutation of protoplasts with diethylsulfate,and its itaconic acid production reached 50.46 g/L,which was 68.08% higher than that of AtUV3-325,and 167.98% higher than that of CICC2433-3.Finally,with the optimization of fermentation process,the final yield of 60.13 g/L of itaconic acid was obtained.(2)Preparation of fermentable sugars of lignocellulose from forestry residues: The bamboo(Sinocalmus affinis)wastes and Oak shells were chosen as raw materials,and the effects of different pretreatment methods on the enzymatic hydrolysis were investigated,and the fed-batch enzymolysis saccharification process was used to prepare high-concentration sugars.Firstly,the effect of alkaline method,acid method,steam explosion pretreatment and the combined pretreatment method on the enzymatic hydrolysis of bamboo was investigated,with the changes of chemical composition,surface morphology and enzymolysis effect as indicators.The results show that the highest delignification rate(51%)can be obtained with 2% NaOH soaking combined with steam explosion,and the enzymatic hydrolysis yield is significantly increased from 24.5% to 47.1%.With SEM,X-ray diffraction and IR analysis,it was found that it has the strongest destructive power to the raw fiber structure,with most of the cellulose vascular bundles are exposed,the lignin molecular structure is destroyed,and the binding sites of cellulase are greatly increased,thereby increasing the enzymatic hydrolysis efficiency.The optimal amount of enzyme loading was 30 FPU/10 CBU/g pretreated samples with gradient enzymolysis.By fed-batch enzymatic hydrolysis process,the final concentration of substrate was 30%,and after 120 hours of enzymolysis,107.7 g/L of glucose,35.81 g/L of xylose,and 7.82 g/L arabinose were finally obtained,with total sugar of 151.33 g/L.Secondly,the effects of alkaline concentration,treatment temperature and treatment time on the enzymatic hydrolysis of acorn shell were investigated.The highest lignin removal rate(39.34%)can be obtained by 2% sodium hydroxide treatment at 121 ?(0.15 MPa)for 60 min,and the monosaccharide yield can reach 494.5 mg/g raw material.The optimal amount of enzyme loading was 20 FPU/20 CBU/1.5 FXU g pretreated sample.By fed-batch enzymatic hydrolysis process,the final concentration of substrate was 30%,and after 120 hours of enzymolysis,121.7 g/L of glucose,11.31 g/L of xylose,and 6.02 g/L of arabinose can be finally obtained,with total sugar of 139.03 g/L.The study provides a high concentration of cheap carbon source for the fermentation of itaconic acid from Aspergillus terreus.(3)Fermentation behavior of A.terreus AtDES3-235 with lignocellulose hydrolyzate to prepare itaconic acid: AtDES3-235 had lower xylose utilization capacity than that of glucose.The high concentration of xylose inhibits the synthesis of itaconic acid.And the optimum substrate concentration of xylose and glucose was 60 g/L and 100 g/L,respectively.When using a mixed carbon source of xylose and glucose,AtDES3-235 preferentially utilized glucose for growth and metabolism,which began to gradually consume xylose when glucose was consumed to a relatively low level.Strains were unable to use the untreated hydrolysate to grow,indicating the severity of inhibition of IA production by Aspergillus terreus.The fermentation process was regulated:buffer used in the hydrolysis of pretreated raw materials inhibited the strain growth and IA production.The substitute of buffer by high purity water had little effect on the release of reducing sugar,and could exclude the inhibitory effect on the acid production;the addition of corn syrup is very important for the bioconversion process of itaconic acid production by the hydrolyzate,which chould improve the tolerance of strain in the hydrolyzate;finally,by adjusting the carbon source concentration of the hydrolyzate,the IA yield from bamboo and oak shell hydrolyzate reached 19.35 g/L and 13.36 g/L,respectively.And with fed-batch fermentation,AtDES3-235 finally synthesized 41.54 g/L IA from bamboo hydrolyzate,69.08% of the fermentation level of pure glucose medium and higher than the level reported in existing literature.Through optimizing the fermentation process,the strain AtDES3-235 can use the bamboo/oak shell hydrolysate to produce IA without detoxification,and with fed-batch fermentation,the IA yield reaches a higher level,which provides a good fermentation strain and new raw materials for the itaconic acid fermentation.