The Study On L-lactic Acid Production By Rhizopus Oryzae From Lignocellulosic Waste

This study focuses on an economical fermentation process for L-lactic acid production from lignocellulosic biomass by Rhizopus oryzae(R. oryzae). First, we studied the glucose/xylose as carbon source for L-lactic acid fermentation, and then tried to make use of the fungal biomass; Second, we attempted to solve the waste treatment problem in the xylo-oligosaccharides industry, we determined the best route to produce the L-lactic acid from the xylo-oligosaccharides manufacturing waste residue; Third, we studied the effects of lignocellulose degradation chemicals on R. oryzae growth, end-products production and key enzyme activities; Fourth, we obtained high yield L-lactic acid was obtained from scale-up fermentation in the rotating fibrous bed bioreactor; Finally, we made an economic evaluation of the process of converting lignocellulosic biomass to the target product L-lactic acid to demonstrate the feasibility of this process.(1) The main factors affecting self-immobilization of R. oryzae for L-lactic acid production from glucose were studied in the present study. The optimum fermentation conditions utilizing glucose for R.oryzae were obtained. The optimal results were as follows: 1.50 g/L(NH4)2SO4,0.15 g/L KH2PO4, 0.375 g/L MgSO4·7H2O and 0.10 g/L ZnSO4·7H2O. Based on the optimum fermentation conditions, the highest conversion rate of L-lactic acid was 0.76 from 80 g/L glucose, the ethanol yield was 0.09 and the biomass yield was 0.05. The specific activity of LDH(f), ADH(f) and ADH(b) was 0.94, 20.07 and 3.62 U/mg, respectively. Furthermore,R.oryzae can utilize the poor carbon source xylose to produce L-lactic acid. The highest conversion rate of L-lactic acid was 0.58 from 15 g/L xylose, the ethanol yield was 0.04 and the biomass yield was 0.10. The specific activity of LDH(f), ADH(f) and ADH(b) was 0.19, 5.98 and 0.89 U/mg, respectively. We found significant positive correlations between LDH and L-lactic acid titer, and ADH and ethanol titer.(2) The chitosan characterization showed that there was not much difference in molecular weight, thermal stability and infrared spectrum analysis between fungal and commodity chitosan. The deacetylation degree(89.4%) was higher in R.oryzae chitosan and the viscosity was lower in R. oryzae chitosan(5.13 mPa·s), the crystallinity was much lower in fungal chitosan with no obvious X-ray diffraction peak.(3) In this study, waste residue from corn cob after xylo-oligosaccharides manufacturing was used as an alternative abundant, renewable, and inexpensive substrate for L-lactic acid production. The conversion yield of cellulose to glucose was 84.32% with a glucose titer of 61.32 g/L, whereas the conversion yield of hemicellulose to xylose was 63.06% with a glucose titer of 15.90 g/L when the substrate loading was 10%. After enzymatic hydrolysis, 59.41 g/L glucose and 15.45 g/L xylose in the hydrolysate were converted to 34.0 g/L L-lactic acid, equivalent to a yield of 0.34 g g-1dry waste residue by R. oryzae in separate hydrolysis and fermentation(SHF) at 10% substrate loading.(4) A high temperature resistant strain R. oryzae NL02 was obtained by atmospheric and room temperature plasma. L-lactic acid, by-product ethanol and the conversion yield was 72.46 g/L, 5.59 g/L and 0.73, respectively, from 100 g/L glucose by R. oryzae NL02 at 40.Simultaneous saccharification and fermentation(SSF) can synchronize enzymatic hydrolysis and microbial fermentation in a single step. A higher L-lactic acid titer(60.29 g/L) and yield(0.60 g/g dry waste residue) were achieved in SSF with 10%(w/v) substrate loading at high temperature 40 by R. oryzae NL02, demonstrating, for the first time, the feasibility of L-lactic acid production from xylo-oligosaccharides manufacturing waste residues. The L-lactic acid titer and yield of SSF was 1.76 times of SHF at 10% substrate loading.(5) Compared with alkali-pretreated corncob, the total degradation product was 8.11 times higher from acid-catalyzed steam-exploded corn stover hydrolysate. For carbohydrate derived inhibitors, the furan compounds, including furfural and 5-hydroxymethyl furfural, were highly toxic at 0.5 and 1 g/L, while formic and acetic acids at less than 4 g/L and levulinic acid even at 10 g/L were not toxic. Among the phenolic compounds at 1 g/L, trans-cinnamic acid and syringaldehyde had the highest toxicity while 3,4-dihydroxybenzoic acid, 4-hydroxybenzoic acid, vanillic acid, 4-hydroxybenzaldehyde, and vanillin showed moderate toxicity, and syringic, ferulic and p-coumaric acids were almost non-toxic to R. oryzae. Although these inhibitors in the corn stover and corn cob hydrolysates were present at concentrations much less than their separately identified toxic levels, L-lactic acid fermentation with the hydrolysates showed a much more inferior performance compared to the control using a synthetic medium without the inhibitors, suggesting synergistic or compounded effects of the lignocellulose-degraded compounds on inhibiting L-lactic acid fermentation by R. oryzae.(6) Based on the morphology of R. oryzae, the SSF process for L-lactic acid production from xylo-oligosaccharides waste residues was also demonstrated in a 5-L stirred-tank bioreactor, although further optimization would be necessary. The L-lactic acid yield was 0.42 g/g dry biomass in rotating fibrous bed bioreactor. For the economic evaluation of 50,000 tons of L-lactic acid production from lignocellulosic biomass needed to achieve scale-up production, it was estimated that the project investment payback period was 3.24 years and the annual profit margins might reach 30.87%. The unit production cost was 2760.14 $/MT MP with the raw materials and utilities accounted for 75.79% and 13.28%, respectively.