Research On Direct Submerged Fermentation Of L-lactic Acid With Immobilized Rhizopus Oryzae By Corn Cob

L-lactic acid has a wide range of application, particularly Poly L-lactic acid（PLA）polymerized with L-lactic acid was found in recent years, which has thebiodegradable property so that expected to replace all kinds of not biodegradableplastic such as PVC, PP etc in the future, and eliminate the "white pollution",therefore, L-lactic acid fermentation has a great application prospect. Corn cob iswaste of the maize industry, at present there only small percentage used forproduction xylose, wine, etc, the majority burned directly as a farm fuel, its valueneeds further development. In this paper, corn cob used as partial substitute for carbonsource and carrier immobilized Rhizopus oryzae to ferment L-Lactic acid, the processconditions were optimized, the relationship between the product accumulation and themorphological changes of the mycelium and carrier was investigated, the theoreticaland practical conclusion will be the foundation for its industrial application. The mainresearch contents and results are as follows:Screening from16Rhizopus oryzae lactic acid producer with the congo red plate,the original strain R-1was obtained. Treated with ultraviolet, the mutants which canutilize corn cob were selected with the corn cob plate as the sole carbon. Oxford Cupcorn cob and bromocresol green agar plate （The modification Oxford Cup method）was established and employed, the mutant R-1-4was obtained, and the method wasverified as efficient screening method which can select high producing mutant usingcorn cob by shaking flask fermentation. The utilization ratio of corn cob as the carbonsource of strain R-1-4directly reached14.9%, more than other high cellulaseactivity strains, suitable for the raw material fermentation of corn cob.As carrier and Raw material, the corn cob ferment L-lactic acid. With L9（34）orthogonal experiment optimization, the concentration of （NH4）2SO₄KH2PO₄MgSO₄ZnSO₄were respectively3g/L0.3g/L0.4g/L0.2g/L, the initial pH was6.0, thefermentation temperature was30℃, the rotating rate was200r/min, after48hfermentation, the accumulation of L-lactic acid was32.23g/L, and the conversion rate of glucose to L-lactic acid reached80.58%. The optimum proportion of corn cob andglucose mixed fermentation was1:4, and the conversion rate of glucose to L-LacticAcid reached82.5%, increased by14.6%in comparison to the pure glucosefermentation. The optimum fineness of the corn cob was40mesh, on this condition,the yield of acid with Rhizopus oryzae R-1-4fermenting the mixed carbon sourceswas34.68g/L, meanwhile, the immobilized mycelial pellets with continuousfermentation ability were formed and theirs diameter were0.5-1.0mm, the averageconversion rate was81.3%after6batches of continuous fermentation.The surface and internal morphology of the mycelial pellets were observed withmicroscope, it showed that, four layers structure was formed which benefit theproduct accumulation, and the mycelia loosed gradually from outside to inside. Thereason of acceleration lactic acid production based on mycelial pellets immobilized bycorn cob was explained microcosmically, and the morphological proof was providedto study the influence of the mycelial morphology during the fermentation process ofRhizopus oryzae. Research on the morphology of corn cob before and afterfermentation with electron microscope, the results show that, unfermented corn cobhas fiber aggregation structure, macromolecular chains arrange orderly with tightstructure, less cracks and holes, and combine strongly with each other. While theregular structure of corn cob is disrupted after fermentation, the number of the holeswith relatively loose structure increased. It can be found obviously that the cellulose isbroken down in the fermentation process, to be used as carbon sources.The fermentation tests were completed in a10L automatic fermentor withRhizopus oryzae R-1-4in the optimal conditions, and obtained the fermentationprocess curve. Analysis of the fermentation process parameters, Logistic equation andLuedeking-Piret equation were chosen as the kinetic models, the batch fermentationdata were fitted by software origin8.1. Three kinetic models were constructed, whichcould reflect the growth of microbe, product formation and substrate consumptionrespectively and fit very well. They could suitably describe the immobilizedfermentation process of Rhizopus oryzae R-1-4, otherwise, provide the theoryfoundation for the magnified production and the optimal controlling.