Optimization Of 3-hydroxypropionic Acid Fermentation Process

3-Hydroxypropionic acid(3-HP)is a precursor that can be easily converted into an array of economically important compounds.As such,3-HP was proposed as one of top 20 value-added platform chemicals by the US Department of Energy(DOE).In recent years,3-HP has garnered much attention due to its economic importance.Despite impressive achievements in this regard,bioproduction of 3-HP remains in its infancy,and most endeavors are still limited to laboratory stage.The current low yield and high cost constrain its commercialization.Hence,development of a cost-effective 3-HP bioprocess is highly desirable.This present study aimed to reduce production cost.First,we investigated whether the mixture of glycerol and glucose were more effective for 3-HP production compared with using glycerol or glucose alone.We also disentangled whether the mixture are beneficial for cell growth under previously reported conditions.Results showed that although the mixture of glycerol and glucose benefited cell growth,the 3-HP level was not significantly improved.To develop a cost-effective bioprocess,the low-cost crude glycerol was used as carbon source and evaluated its influences on 3-HP biosynthesis.Results showed that crude glycerol was not suitable for bacterial growth relative to pure glycerol.Collectively,pure instead of crude glycerol is an ideal carbon source for 3-HP production.Second,the yeast extract concentration was optimized to improve 3-HP production.In shake flask cultivation,the strain produced 10.38 g/L 3-HP when 53 g/L yeast extract concentration was added into medium.In addition,three low-cost organic nitrogen sources including soybean meal,corn syrup and cottonseed meal were investigated for their impacts on 3-HP formation.Results showed that 3-HP peaked when 5.3 g/L soybean meal or 4.89 g/L corn flour was added into shake flask,and both organic nitrogen sources were shown to be suitable for 3-HP production.Surprisingly,when cottonseed meal was used as nitrogen source,bacterial growth was obviously halted.As a result,only 2.5 g/L 3-HP was produced.It is clear that cottonseed meal is not suitable for fermentation production of 3-HP.Third,based on above shake flask experiments,bioreactor cultivation was carried out to further determine the effects of different nitrogen sources on 3-HP production.The results showed that this strain produced 113 g/L 3-HP when 53 g/L yeast extract was added into medium.To the best of our knowledge,this is the highest 3-HP titer reported so far.Moreover,when 9 g/L soybean meal or corn syrup was added into medium,this strain produced 91 g/L or 90 g/L 3-HP,respectively.Interestingly,when the same amount of yeast extract was used,the strain produced 93 g/L 3-HP.Since yeast extract is more expensive than corn syrup and corn syrup,the latter two nitrogen sources are selected for further studies.Finally,reverse transcription analysis of key enzyme genes in the glycerol pathways was performed to determine how nitrogen sources affect the production of 3-HP.In particular,we analyzed the expression of the aldehyde dehydrogenase puuC which directly catalyzes the formation of 3-HP.The results demonstrated that nitrogen sources contributed a lot to 3-HP biosynthesis.Now that soybean meal is the most effective and inexpensive nitrogen source for 3-HP production,it was subjected to further fermentation optimization.The optimum fermentation conditions concluded:pH at 7.5,temperature at 30℃,rotation speed at 100 rpm,and dissolved oxygen at 1.2 vvm.In these conditions,the strain produced 93 g/L 3-HP.Although 3-HP level was not significantly enhanced,the energy was less consumed compared with previous experimental result.