The Effect Of Lactococcus Lactis Aerobic Respiration By PH On Fermentation Broth

Lactococcus lactis is widely applied in the manufacture of fermented dairy products.It is also used as an excellent cell factory to express heterologous genes and live vector for the mucosal delivery of therapeutic proteins.Cell density of starter culture is a key factor for determining starter performance,increasing yields and reducing production cost,thus,high-cell-density cultivation of L.lactis is very important.It belongs to facultative anaerobic microbes which is lack of complete respiration chain.It generates energy only via fermentation.It has been indicated that in the presence of hemin,L.lactis can synthesize key enzymes involved in aerobic respiration,and form a complete electron transfer chains,thus fermentation metabolism shifts to aerobic respiration,which results in more superior biomass and survivability.pH has very important influences on cell physiology such as nutrient uptake and intracellular enzymes activities.In the industrial processes,pH is a common fermentation control parameters,thus it is of great practical significance for obtaining high-cell-density culture to study the optimal pH value at which respiration efficiency is high.This paper studied the influence of culture pH on biomass yield and respiration efficiency by determining the biomass and residual reducing concentration in fermentation liquid of L.lactis NZ9000(pNZ8148)under different conditions varied in culture pH,aeration and addition of hemin.A spectrophotometic method for estimating intracellular hemin was established,and the relationship between culture pH and intracellular hemin accumulation was studies.The main results are as follows.1.The biomass of L.lactis NZ9000(pNZ8148)cells cultured in the fermentor supplied continuously with dissolved O2(the A conditions)was only 40 % of that of the cells incubated under the HA conditions(4 ?g hemin/m L plus O2).The growth rate of the HA cells in the early stage of the logarithm phase was already lower than that of the A cells,and the logarithm period of the former was longer than the latter for 3 hours.These demonstrated that L.lactis NZ9000(pNZ8148)can undergo respiration during the whole cultivation,instead of initial fermentation then turning to respiration.The pH values of the HA and A cultures both constantly decreased;although the declining velocity of culture pH of the former was obviously higher than the latter,the terminal pH values were almost the same,which indicated that the inhibitory effect induced by acid stress still existed,but it may be reduced by respiration to a certain extent.pH controlled at 6.5 resulted in a markedly improvedbiomass in the respiratory culture;the maximum biomass yield(in g CDW per g reducing sugar)only reached 67 % of the HPA6.5 cultures.This implied that under acid stress eliminating conditions respiring L.lactis NZ9000(pNZ8148)cells has ability to conversing more reducing sugar to biomass than fermentative growth.2.L.lactis NZ9000(pNZ8148)cells were cultivated under the HP and HPA conditions with pH controlled at 5,5,5 and 6.The effect of culture pH on respiratory benefit was observed in the following order: pH 5>5.5>6>6.5 because the biomass yield under respiration-permissive conditions increased by 244,180,94,and 50 %,respectively,compared with the corresponding fermentative conditions when pH was controlled at 5,5.5,6,and 6.5.3.The absorbance(A380)relations were plotted against 0.1–4?g hemin/mL and fitted with a straight line determined by linear regression.Hence,a sensitive and accurate quantification method was established to detect intracellular hemin in an increased biomass of L.lactis NZ9000(pNZ8148)culture(OD600>2).4.Intracellular hemin concentrations of L.lactis NZ9000(pNZ8148)cells grown under the HPA conditions with pH controlled at 6.5,6,5,5 and 5 were determined.culture pH affected the hemin uptake capacity of L.lactis NZ9000(pNZ8148)in the following order: 5.5>6>5>6.5.Analysis of the relationship between intracellular hemin amounts,biomass and respiration efficiency revealed that pH 5.5 could promote hemin to accumulate in the L.lactis NZ9000(pNZ8148)cells,then enhance respiration;as a result,a relatively high biomass was obtained.