Study Of A Novel Immobilzation Matrix In L-Lactic Acid Production By Rhizopus Oryzae

Recently, L-lactic acid has attracted a lot of attentions for its use as the starting material in the synthesis of polylactic acid, which can be used for manufacture of biodegradable materials. Current industrial lactic acid fermentations use homolactic acid bacteria, which have high lactic acid yield and productivity but are fastidious and require complex nutrients for fermentation. In contrast, some filamentous fungi such as Rhizopus oryzae and Rhizopus arrhizus can grow in simple media with minimal nutrients to produce L-lactic acid from various carbohydrates, including glucose, xylose and starch. Besides the simple medium cost, the fungal fermentation also has the advantage of producing L-lactic acid with a high optical purity of greater than 99% that is easier to purify from the simple medium used in the fermentation. However, filamentous fungi are difficult to use in large-scale industrial fermentation because of their complex morphology, substrate cost and process engineering instability in scaling-up.A new support matrix inspired by honeycomb was developed for cell immobilization to control fungal morphology and enhance mass transfer in bioreactor for lactic acid production by Rhizopus oryzae. The immobilization matrix composed of asterisk-shaped fibrous matrices in a honeycomb configuration provided high surface areas for cell attachment and biofilm growth. The feasibility, scaling-up potential, stability and scope of application of the matrix were all investigated.First, in immobilization and fermentation kinetics study, lag phase of germination process (tβ) and 50% immobilization time (λ) were determined by germination plot. Compared to to other environmental factors such as temperature, pH, and nutrients, the rotational speed did not strongly influence R. oryzae spore germination. The cell adsorption kinetics was found to follow the first-order reaction kinetics and can be represented by ln( Ct /Co)= ?kt, The adsorption rate constant k can be estimated from the slope of the semi-logarithmic plot of C vs. t and was found to be 0.12±0.04 h-1 for the initial 4-h period and 0.50±0.07 h-1 for the subsequent period after spore had germinated.Furthermore, the immobilized fermentation using the novel matrix was optimized both in environmental factors and medium compositions. The optimal culture setting was as following: rotational speed 180 r/min, matrix diameter 2 cm and three matrices in each flask, initial spore concentration 1×106/ml. Uniform design was carried out in medium optimization. The highest L-lactic acid production was achieved when the medium consisted of : 80g/L glucose, 0.15g/L urea, 0.5g/L KH2PO4 , 0.2g/L MgSO4.7H2O,0.04 g/L ZnSO4.7H2O,0.006 g/L FeSO4.5H2O,40g/L CaCO3 added every 12 hours.Then, the novel matrix was compared to calcium alginate gel, polyvinyl alcohol gel and loofa sponge in immobilized fermentation by R.oryzae. In batch fermentation, novel matrix obtained better morphological control and L-lactic acid production and other methods. Moreover, it showed better stability and producing ability in long-term fermentation, while both calcium alginate gel and polyvinyl alcohol gel tended to abrupt after relatively short period.In study on preference of carbon assimilation of R.oryzae using this matrix, glucose, corn starch (soluble and in soluble), corn cob hydrolysate and xylose were investigated. Corn starch was proved to be similar with glucose in L-lactic acid production, while corn cob hydrolysate was not favorable because cellobiose and xylose accumulation in fermentation limited cell activity of R.oryzae. The long-term fermentation using glucose/xylose proved that R.oryzae preferred glucose to xylose and the potential of xylose could be explored by assistant of glucose.At last, matrices were united in 350ml and 3.5 L column bioreactors to testify its scaling-up feasability and process parameters. In 350 ml column bioreactor, the best culture conditon was: 120g/L glucose, 106 spore /ml, pH 6.0 by adding 10 M NaOH solution. Volumetric oxygen transfer rate coefficient (kLa) was found to be linearly related with superficial air velocity (Vs). Compared to oxygen transfer rate coefficient, oxygen transfer rate was more reliable as the scaling-up key parameter since it included liquid height and air pressure in the process.