Anaerobic Digestion Of Waste Pichia Pastoris In Couple With Enhanced/Cleaner Butyric Acid And Butanol Productions

Recombinant Pichia pastoris is an effectivly system for the expression of heterologous proteins,such as biological enzymes and pharmaceutical proteins.semi-solid waste Pichia pastoris is a typical industrialized waste biomass with huge treating difficulty.On the other hand,waste P.pastoris contains abundant valuable polysaccharides and proteins,the waste amount recirculation and reduction are expected by digesting waste P.pastoris using anaerobic fermentation technique.Butyric acid/butanol are important platform chemicals/biofuel which could be produced by fermentation methods.Those fermentation processes require sufficient amino acids and carbohydrates as nitrogen and carbon sources which limit their industrial applications.The traditional fermentations suffer many problems such as high prices of the raw materials,complicated operation procedure,lower products titers or quality,poor economic benefits,etc.In this thesis,a novel fermentation technology,anaerobic digestion of waste Pichia pastoris in couple with enhanced/cleaner butyric acid and butanol productions,was proposed and implemented.The main results are summarized as follows:（1）The expensive organic nitrogen source required for butyric acid fermentation was replaced by using the waste P.pastoris suspension.The butyric acid fermentation strategy,which use corn starch medium(80 g-corn·L^-1)as the initial starting medium,followed by adding 18-28 g-DCW·L^-1waste yeast suspension and concentrated glucose solution during fermentations,was developed.Final butyric acid concentration and ratio of butyric acid over total acid（B/TA）reached a high level of 45 g·L^-1 and 90%,respectively.This strategy can anaerobic digeste amino acids generated from the waste yeast suspension,promote cells growth rate and concentration,and thus improve butyric acid fermentation performance indirectly.（2）Traditional butyric acid/butanol fermentations suffer some obstacles to implement,such as using high-cost raw materials（using corn starch to start/initiate the fermentations）and biological enzymes for treating the raw materials,as well as the complicated pre-treatment operations.A novel stategy which directly mixing Na OH pretreated waste yeast suspension with glucose as starting medium(5.6 g-DCW·L^-1 waste yeast suspension,about 4.0 g/L SO₄^2-)to replace corn-starch based“starting medium”could tolerate Clostridium tyrobutyricum against high SO₄^2-environment,and thus the fermentations could be directly initiated.At about24 h,when Clostridium tyrobutyricum grows to certain degree,and it could tolerate a higher SO₄^2-environment.At this moment,26.3 g-DCW·L^-1 waste yeast suspension（step-wise）and the concentrated glucose solution（consecuticely）were added into the broth during fermentations.Final butyric acid concentration reached 51-54 g·L^-1,which was 160%higher than that of the control(80 g·L^-1 corn starch medium).Butyric acid accounted for 98%of the total acid.70%amino acids and 66%oligosaccharides originated from waste yeast were utilized or consumed,while the apparent reduction rate of single batch fermentation waste yeast was only 49%.A mathematical model for multiple reuses of butyric acid fermentation waste yeast was constructed and the results shows that when the total number of fermentation units is 4（N=4）,the apparent reduction rate of waste yeast can reach 97%.（3）Bio-butanol is an efficient liquid fuel.The similar starting and feeding media using waste yeast and concentrated glucose used by butyrate fermentation were also applied for butanol fermentation(2.8 g-DCW·L^-1 initial waste yeast suspension+25.6 g-DCW·L^-1 waste yeast suspension during fermentations),the final butanol concentration reached 11.5 g·L^-1.The waste yeast apparent reduction rate in single fermentation batch can reach 71%;the degradation rate of protein is 82%;the utilization rate of polysaccharide degradation is 56%.In butanol fermentation,Clostridium acetobutylicum could utilize more oligosaccharides of waste yeast to convert into ABE products and small molecular substances such as CO₂.（4）At present,butanol fermentation products with high B/A ratio are generally regarded as high-quality butanol fermentation products.Butyric acid is an important precursor for butanol synthesis in ABE fermentation.Utilizing butyric acid fermentation supertanant（BFS）/glucose as co-substrate would efficiently produce butanol featured with high B/A ratio and improve the quality of ABE fermentation products.Compared with the traditional butanol fermentation,the butanol concentration remained at a higher level of 12.7-12.8 g·L^-1,butyric acid/glucose mass consumption ratio of 37%-53%and the B/A was greatly increased from 2.0to 4.4-5.0.The ABE fermentation system effectively utilized butyric acid,oligosaccharides and amino acids in BFS,and the NADH utilization efficiency,butanol synthesis and the B/A ratio were significantly improved.The system can also save the raw material and operating costs and realize the diversity of fermented products.（5）Economic analysis of the butyric acid/butanol fermentation systems using waste yeast as raw material was evaluated.The results showed that consuming 1 t-WCW waste Pichia pastoris,￥2,100-￥3,500?（t-WCW）^-1 gross-profit in butyric acid fermentation and￥2,400-￥2,800?（t-WCW）^-1 gross-profit in butanol fermentation could be achieved,leading to with high economic benefits.