Process Optimization And Mechanism Of Microbial Protein Production By Co-culture Of Methane Oxidizing Bacteria And Hydrogen Oxidizing Bacteria

With the explosive growth of the world population and the rapid increase in the level of urbanization and industrialization,the safe and efficient supply of food and protein becomes more and more critical.It is not sustainable to increase traditional agricultural acreage blindly,and new method of high-quality protein supply and harmless treatment of waste need to be developed.In this study,microbial protein production using methane oxidizing bacteria and hydrogen oxidizing bacteria was investigated.The metabolic growth of methane oxidizing bacteria and hydrogen oxidizing bacteria under two phase culture was firstly investigated.Then the carbon and nitrogen fixation capacity and microbial protein production under co-culture of methane oxidizing bacteria and hydrogen oxidizing bacteria were investigated.Subsequently,the feasibility of microbial protein production by methane oxidizing bacteria and hydrogen oxidizing bacteria in actual wastewater was verified.Finally,the coupled culture of methane oxidizing bacteria and hydrogen oxidizing bacteria was carried out in real wastewater.（1）A mixed flora dominated by methane oxidizing bacteria and hydrogen oxidizing bacteria was domesticated.The growth of methane oxidizing bacteria and hydrogen oxidizing bacteria in two-phase culture was investigated.The two-phase culture was performed with the methane-oxidizing bacteria system as the first phase,and the outgas went to the hydrogen oxidizing bacteria system（i.e.,the second phase）for further protein synthesis.The results showed that CH₄in the first phase outgas did not significantly promote or inhibit the growth and metabolism of the second phase hydrogen oxidizing bacteria with<10%change of cell dry weight and MP content relative to no CH₄addition.The H₂/CO₂（m L/m L）was 5.42 and 13.83 for single-phase and second-phase hydrogen oxidizing bacteria,respectively,indicating that the energy required to fix a unit of CO₂was high for the second phase hydrogen oxidizing bacteria;the microbial protein yields were 1.71 g/L and 0.92g/L,respectively,and the carbon utilization efficiencies Y_CDW/Cwere 0.56g CDW/g C,2.18 g CDW/g C,respectively.It indicated the percentage of carbon conversion to cell dry weight was greater for the second phase hydrogen oxidizing bacteria.As shown by the microbial community analysis,the dominant genera in the single-phase were Hydrogenophaga（75%）and Xanthobacter（9%）,and the dominant bacteria in the second phase were mainly Aquamicrobium（34%）and Defluviimonas（15%）.Therefore,although the protein production of hydrogen oxidizing bacteria in two-phase culture was low,the carbon utilization efficiency was high.（2）The advantages of carbon fixation and microbial protein production of the flora in co-culture were investigated compared to separate culture.The co-cultivation process involves the cultivation of methane oxidizing bacteria and hydrogen oxidizing bacteria in the same reactor.The co-culture system had a complete metabolism of H₂,CH₄,O₂and CO₂consumption,and the total gas consumption,especially the total carbon fixation,was close to the sum of monoculture hydrogen oxidizing bacteria and methane oxidizing bacteria.In terms of the final yield of the bacteria obtained,the dry weight of cell in co-culture reached 0.85 g/L,significantly higher than that of monoculture hydrogen oxidizing bacteria and methane oxidizing bacteria,exceeding the sum of the cell dry weight of both by 0.71 g/L.The amino acid yield reached 311.0 mg/L which was1.3 times higher than the sum of 233.2 mg/L in monoculture.The co-culture had the highest efficiency of ammonium to cell dry weight conversion efficiency of 10.87 g CDW/g N.Community analysis showed that the co-cultured flora were mainly Methylocystis（39%）,Hydrogenophaga（8%）.Therefore,co-culture had great advantages compared to monoculture and was an ideal way to achieve carbon fixation and microbial protein synthesis.（3）The growth of methane oxidizing bacteria and hydrogen oxidizing bacteria in monoculture in real wastewater was investigated.The methane oxidizing bacteria was more active and adaptable,with carbon consumption rate of 30 mg C/d and ammonium nitrogen consumption rate of 11 mg N/d,producing cell dry weight of 1.2 g/L and crude protein content of 36.7%.In contrast,the hydrogen oxidizing bacteria were inhibited in actual wastewater.After doubling the dilution of the wastewater,the inhibition of hydrogen oxidation bacteria was relieved,and the time to degrade ammonium nitrogen in the wastewater was shortened to 8 days at the earliest.When S was added to the medium,the change in the rate of degradation of ammonium nitrogen was<7%compared to that without the addition of element,and the change in final cell dry weight was<15%,indicating that the insignificant effect of S in system.When Mg,K and P were added to the wastewater,the rate of ammonium nitrogen degradation was slower when Mg and K were added than that without the addition of element,and the change in final cell dry weight was<7%,excluding the effect of Mg and K.However,the rate of ammonium nitrogen degradation was 17%higher than that without addition when P was added,and more than 2.0 g/L,66%higher than without addition biomass,was obtained.The ammonium to cell dry weight conversion capacity was 18.57 g CDW/g N and carbon to cell dry weight conversion capacity was 1.24 g CDW/g C with addition of P.P addition promoted the flow of nitrogen and carbon to crude protein synthesis.In addition,the three Mg,K and P did not have a synergistic effect yet.At the end of the experiment,the variation of rare earth element concentration with the addition of Mg+K+P was analyzed,and a total of 16 rare earth elements were detected.It was found that the colony was absorbing and enriching for rare earth elements,and the recovery of rare earth elements were all in the range of 80-130%.In conclusion,methane oxidizing bacteria and hydrogen oxidizing bacteria could grow and metabolize in wastewater,and the possible factor inhibiting the growth of hydrogen-oxidizing bacteria was the lack of P.（4）The effects of different coupled systems on microbial protein production by methane oxidizing bacteria and hydrogen oxidizing bacteria in actual wastewater were investigated.The coupled systems were the two-phase culture mode as well as the co-culture mode.In both modes of the coupled system,carbon reduction was achieved,with carbon reduction of 17 mg C/d and 40 mg C/d achieved in two-phase culture and co-culture,respectively,and ammonium recovery rate of 10mg N/L and 12 mg N/L,respectively.Protein production of 0.6 g/L was achieved in two-phase culture at lower substrate demand,which was nearly one time higher than that in co-culture mode of 0.3 g/L.The concentration of rare earth elements in the co-culture was analyzed at the end of the experiment,and a total of 16 rare earth elements were detected,and their enrichment rates were concentrated in the range of 30-70%,with the highest enrichment rate reaching 83%of cerium,showing the strong enrichment effect of microorganisms on rare earth elements.Consequently,under the coupled system,two-phase culture was the optimal culture for microbial protein production by methane oxidizing bacteria and hydrogen oxidizing bacteria in real wastewater.