Optimization Of Media For Hansenula Polymorpha By Vitamins And Amino Acids

As a common industrial interested organism,methyltrophic yeast Hansenula polymorpha can utilize both glucose and methanol as the sole carbon sources for cell growth and product biosynthesis.Therefore,H.polymorpha is suitable for industrial production of high value-added chemicals due to its fast growth,wide substrate spectrum,high temperature tolerance,and easy cultivation.Here,we optimized synthetic mediums containing methanol and glucose as the sole carbon sources to maintain the basic growth of H.polymorpha.On this basis,types and concentrations of multiple vitamins and amino acids were optimized by using single factor experiment and response surface method.Our goal is to achieve faster and better growth of H.polymorpha cells when supplementing as few nutritions as possible,and an enhanced cell biomass with a decreased total costs may lay a good foundation for further applications of H.polymorpha in industrial application.Main results are listed as follows:?1?While using methanol as the sole carbon source,the optimized medium contained?NH₄?₂SO₄ 2.5 g/L,KH₂PO₄ 14.4 g/L,MgSO₄·7H₂O 0.5 g/L,trace-metal 2 ml/L,leucine 60mg/L,methanol 10 g/L,biotin 0.005 mg/L and thiamin 1.04 mg/L.Under the optimal conditions,at the stable phase of 80 h,maximum biomass was achieved up to 10(OD₆₀₀),which was 2.5 times higher than that in initial basis medium.?2?The optimized medium with glucose as carbon source is listed as follows:?NH₄?₂SO₄2.5 g/L,KH₂PO₄ 14.4 g/L,MgSO₄·7H₂O 0.5 g/L,trace-metal 2 ml/L,leucine 60 mg/L;glucose 20 g/L,biotin 0.005 mg/L,methionine 40 mg/L,glutamic acid 200 mg/L,and aspartic acid 200 mg/L.After 60 h of fermentation,the biomass reached the maximum(OD₆₀₀=20²4),which was 7-8 times than that in initial basis medium.?3?The promotion of vitamins and amino acids to cell growth had been proved by an enhanced intracellular activities of the specific enzymes like pyruvate dehydrogenase and pyruvate carboxylase.Results indicated that biotin and thiamin dramatically increase the activities of both pyruvate dehydrogenase and pyruvate carboxylase in the optimized medium with methanol,which was in agreement with the hypothesis that both biotin and thiamin acts as coenzymes to promote cell growth.Similarly,the activity of pyruvate carboxylase was also significantly increased by biotin and amino acids in the medium with glucose as carbon source.