Effect Of Microaeration On Fermentation By Kluyveromyces Marxianus From Lignocellulosic Hydrolysates

The production of fuel ethanol by lignocellulosic materials has been paid more and more attention,due to the wide availability of raw materials,good renewability and less pollution.However,the inhibitory effect of toxic by-products mainly deriving from the pretreatment of lignocellulosic biomass on fermentative microorganisms is one of the limiting factors hindering the industrial development of bioethanol.In this paper,firstly,direct evolution was used to obtain yeasts with high resistance to multiple inhibitors.Secondly,microaeration strategies were utilized to alleviate the adverse effects of lignocellulose-derived inhibitors on the yeasts.The main results are listed as follows:(1)Direct evolution was applied in Kluyveromyces marxianus 1727-5 in the presence of dilute-acid hydrolysates of corn stalks,and a strain named K.marxianus 1727-5-CS with a slight improvement in tolerance to acetic acid was obtained.(2)In the presence of multiple inhibtors including formic acid,acetic acid,furfural and5-HMF,the effects of constant-rate aeration by pumping air at the rates of 0.1,0.2,0.5 or 1vvm on fermentation with glucose,xylose and their mixture as the carbon source,respectively,were studied.Aeration significantly improved fermentative performance of the strain under the stress of multiple lignocellulose-derived inhibitors,such as enhancing the rate of xylose consumption,increasing xylitol yield,and shortening the fermentation time.When the aeration rate was increased from 0.1 to 0.5 vvm,the xylitol productivity and yield of xylitol increased,achieving the highest(0.22 g/L/h and 0.7 g/g)at 0.5 vvm.In spite of a slight improvement of xylose fermentation by enhancing the initial biomass concentration,it is not the main reason for the well-deserved parameters compared with aeration.Under aeration condition,glucose consumption was enhanced with a similar ethanol yield(0.41±0.01 g/g),and a significantly reduced glycerol production,which also indicated that aeration alleviated the adverse effects of inhibitors on cells.For co-fermentation of high or low concentrations of glucose and xylose,the xylose was barely consumed before glucose was exhausted under conditions without aeration.In contrast,the fermentation of xylose was not affected by the glucose under 0.5 vvm,and the residual xylose was decreased to 3.6 g/L and 11.1 g/L from17.1 g/L and 28.5 g/L compared with the control.In addition,it was found that the transcription levels of KmTPX1,one of the key genes defending the damages by reactive oxygen species,was over three times higher than the control at 0.2 vvm,which may be the reason for the enhancement of tolerance to multiple lignocellulose-derived inhibitors.(3)In the presence of lignocellulose-derived inhibitors,fermentation with glucose,xylose and their mixture as the carbon sources were also conducted by oxidation-reduction potential(ORP)control.And the fermentation with corn stalk hydrolysates was also investigated under different aeration strategies.The damages to cell growth and metabolism that were caused by multiple inhibitory stresses and anaerobic condition may be effectively improved under the ORP control.With the increase of default ORP values,aeration volumes increased,cell viability enhanced,and glucose and xylose metabolism accelerated.For co-fermentation of glucose and xylose,when the ORP was controlled at-150 mV and-110 m V,the stagnation of xylose consumption were effectively removed in the late fermentation stage.The xylitol yield reached the highest(0.40 g/g)at-150 mv,and the ethanol yield was as high as 0.42 g/g.(4)Aeration strategies also effectively alleviated the adverse effects of corn stalk-derived inhibitors on the yeast,and improved its fermentative performance.It was found that the yields of ethanol and xylitol achieved 0.42 g/g and 0.13 g/g at 0.5 vvm,compared with those of 0.44 g/g and 0.48 g/g,respectively,when the ORP was controlled at-100 mV.And the aeration volume of ORP control was 10% less than that at 0.5 vvm.Therefore,considering the ethanol production from corn stalk hydrolysates,controlling ORP at-100 mV is supposed to be an effective and efficient approach.In conclusion,the inhibitor-resistance and fermentative performance of yeast was improved by both constant-rate and ORP-controlled aeration,which has also been proved as a simple and feasible method with the promising potential for industrial applications in cellulosic ethanol.