The Study Of Pretreatment Of Laminaria Japonica Biomass And Ethanol Transformation Of Mannitol

The third generation bioethanol which is made from marine biomass has become a hotspot that the whole world cares. To make use of algae to ferment and produce bioethanol, we need to pretreat algae, optimize pretreatment and fermentation conditions, and screen superior strain. At present, more researches focus on algae ingredients extraction, however, researches with aim to ferment carbohydrate in algae and produce ethanol were limited. In this study, firstly, we optimized pretreatment conditions of Laminaria japonica; secondly, we screened thermotolerant strains which could utilize mannitol in L.japonica and ferment to produce ethanol; thirdly, we studied and optimized fermentation metabolism conditions. The main research results of this paper are shown below:1. Optimization of L.japonica pretreatment conditionsWe firstly analysed the chemical compositions of L.japonica, and the result showed that its moisture content was 5.6%, ash 12.6%, carbohydrate ingredients that could be fermented were respectively:mannitol 23.1%, alginate 25.6%, corase fiber 8.0%, reducing sugar 0.2%. The conditions with acid solution pretreating L.japonica were optimized by means of single factor experiment and response surface analysis, using mannitol as response value, the optimum pretreatment conditions of L.japonica were:0.11 mol/L hydrochloric acid,60 min,111℃, the actual concentration of mannitol could reach 20.94 g/L; 0.066 mol/L sulphuric acid,60 min,108℃, the actual concentration of mannitol could reach 20.45 g/L. L.japonica residue pretreated by hydrochloric acid was extracted by means of heating method, and the yield of sodium alginate could reach 0.342 g/g, this result showed that heating method was better than cold method, and hydrochloric acid pretreatment method had more advantage than sulphuric acid pretreatment method.2. The screening of strains which could utilize mannitol to ferment ethanolThe thermotolerant yeasts K.marxianus S16、K.marxianus K213、K. marxianus L27、Ogataea sp. WXT3 screened by our laboratory and S. cerevisiae were used as original strain, and mannitol was used as carbon source, only Ogataea sp. WXT3 could make use of mannitol to produce ethanol under the condition of both organic and inorganic nitrogensource, and the yield of ethanol was 0.18 g/g mannitol. Thermophilic anaerobic Thermoanaerobacterium calidiforntis Rxl screened by our laboratory could grow on the mannitol, and the concentration of ethanol could reach 2.0 g/L, higher than Ogataea sp. WXT3 with 0.7 g/L, in addition, both growth rate and fermentation temperature of T.calidiforntis Rxl were higher than Ogataea sp. WXT3, so we choose T.calidiforntis Rxl to go on with the separate hydrolysis and fermentation.3. Characteristics study of L.japonica hydrolysate fermentation by thermotolerant strainsUsing mannitol or hydrolysate of L.japonica as substrate, we studied the effects of T.calidiforntis Rxl fermentation temperature, pH, concentration of substrate on yield of ethanol. The level central values of each factor had been ascertained as 55℃, pH 7.0, concentration of substrate 7.5 g/L by means of single factor experiment. Yield of ethanol was respectively 2.07 g/L and 1.91 g/L, and conversion rate 0.45 g/g and 0.43 g/g under the condition of statically fermentation; yield of ethanol was respectively 2.74 g/L and 2.36 g/L, and conversion rate 0.41 g/g and 0.40 g/g under the condition of waveringly fermentation, showing waveringly fermentation could remarkably raise ethanol production. The optimal fermentation conditions, which was ascertained by orthogonal test, had been determined respectively as temperature 55℃, pH 7.0, concentration of mannitol 10 g/L, concentration of ethanol 3.65 g/L, increasing 23.3% in comparion with the ethanol production without optimization; 50℃, pH 7.0, concentration of L.japonica hydrolysate 7.5 g/L, concentration of ethanol 3.65 g/L, increasing 29.6% compared to the ethanol production without optimization.4. The research of T.calidiforntis Rxl metabolizing acetetateIn this study, by analyzing metabolism of T.calidiforntis Rxl, we suggested that microorganism had metabolic pathway to make use of acetic acid and produce ethanol. With mannitol as carbon source, the addition of exogenetic acetic acid into culture medium could improve the T.calidiforntis Rxl substrate utilization rate, biomass of strains and yield of ethanol, meanwhile, decrease the concentration of acetic acid in fermentation liquid. However, with glucose as carbon source, there was not this phenomenon. This indicated that mannitol could activate acetic acid metabolic pathway of T.calidiforntis Rxl. NMR results proved that T.calidiforntis Rx1 could transform acetic acid into ethanol and lactic acid.