Reduction of foaming and enhancement of ascomycin production in rational Streptomyces hygroscopicus fermentation☆

Xing Xin ,Haishan Qi,Jianping Wen ,2,*,Xiaoqiang Jia ,2,Yunlin Chen

1 Department of Biological Engineering,School of Chemical Engineering and Technology,Tianjin University,Tianjin 300072,China

2 Key Laboratory of Systems Bioengineering,Ministry of Education,Tianjin 300072,China

3 School of Science,Beijing Jiaotong University,Beijing 100044,China

Keywords:Acetate accumulation Fermentation optimization Ascomycin Foaming Oxygen deficiency

ABSTRACT Foaming reduces the working volume and limits the biosynthesis of macrolide immunosuppressant ascomycin(FK520)in the batch fermentation process of Streptomyces hygroscopicus FS-35 in a 7.5 L bioreactor.To find the relation between FK520 production and foaming,effects of 10 fermentation parameters including organic acids and membrane permeability were investigated.The results suggest that acetate accumulation caused by short period oxygen deficiency and fast consumption of glucose is the reason for increased foaming and declined FK520 production.Therefore,a fed-batch fermentation strategy was developed to reduce the accumulation of acetate.After optimization,the maximum acetate concentration dropped from 320 mg·L-1 to 157 mg·L-1,decreased by 50.8%,and the maximum foam height reduced from 5.32 cm to 3.74 cm,decreased by 29.7%,while the maximum FK520 production increased from 375 mg·L-1 to 421 mg·L-1,improved by 12%.

1.Introduction

Foaming is harmful to fermentation processes[1,2]:lowering the working volume of bioreactor and leading to over flow,contamination of fermentation broth,and even strain mutations.Traditional consideration for foaming control is to improve fermentation equipment or add chemical antifoams,so various new antifoams and bioreactors have been developed[1,3–6].These methods suppress foaming effectively but bring some negative effects on fermentation process,such as increasing fermentation costs,suppressing cell growth[1,7],and making the downstream separation of targeted compounds more difficult[2,8].To understand the foaming mechanism in fermentation process,more researchers focus on the effects of cell metabolism and phenotypes on foaming.For example,Patnaik et al.[9]found that the efflux of L-tyr facilitated foam formation,Brey et al.[10]explored the effect of proteinase A on foaming,and Shimoi et al.[11]knocked out the awa1 gene of sake yeast,then efficiently controlled the foaming during fermentation.According to these investigations,the metabolic activity of cells is important for foaming behavior,so it is necessary to analyze the components in the fermentation broth to reveal the metabolic changes of strains with foaming behavior.It has been proved[12]that acetate produced in cell metabolism could affect the membrane permeability apparently,causing a leakage of intracellular metabolites,which may facilitate foaming[7].

In the fermentation study from shake flask to a 7.5 L bioreactor,ascomycin(FK520)production by Streptomyces hygroscopicus FS-35 in the bioreactor was much lower.As the FK520 production rate reduced in the middle stationary phase(96–132 h),the foaming increased.Two traditional approaches were used:installing an antifoaming paddle and adding 4 different antifoams in the fermentation broth separately.Although foaming was effectively controlled,the production of FK520 was not improved.The addition of chemical antifoams even suppressed FK520 production further.Therefore,foaming may reduce the working volume of bioreactor but may not be the direct reason for declined FK520 production in this study.If we know the underlying causes for increasing foaming,it is possible to understand the reasons for declined FK520 production and make corresponding improvement for FK520 biosynthesis.

In present study,dynamic changes of 10 fermentation parameters,including organic acids and membrane permeability,are determined.The effect of each parameter on foaming is assessed.The results suggest that acetate accumulation may be the reason for increased foaming and declined FK520 production.Thus a preliminary fed-batch fermentation mode is developed,reducing acetate accumulation and foam height,while improving FK520 production.

2.Materials and Methods

2.1.Strain,mediums,and shake flask culture conditions

FK520 producing strain used in this study was S.hygroscopicus FS-35,a mutant strain of S.hygroscopicus ATCC 14891[13].The slant and seed medium were prepared as reported[13].The fermentation medium contains(g·L-1)starch 20,dextrin 40,yeast powder7,peptone 5,corn steep liquor2,K2HPO40.5,MnSO40.5,(NH4)2SO41.5,CaCO31,and MgSO41,as well as Tween 80 0.07%(by volume)and soybean oil 11 ml·L-1.The original pH values of fermentation media were maintained at 6.7 by the addition of 5 mol·L-1NaOH.Spore solution(1 × 107spores·ml-1)was added to 250 ml shake flask containing 40 ml seed medium with a 5%(by volume)inoculum,shaking(200 r·min-1)at28 °C for 48 h to obtain the seed.

2.2.Bioreactor culture conditions

The working volume of 7.5 L bioreactor(NBS BioFlo 110,USA)is 4.4 L with a 10%inoculum.The batch fermentation was lasted for 168 h at 28°C.The aeration rate and stirring speed were obtained from previous optimization experiments.The aeration rate was maintained at 1.5 vvm.Dissolved oxygen(DO)was maintained at 50%in lag phase(0–24 h)and 30%in early log phase(24–48 h),then maintained at 10%by setting the maximum stirring speed at 500 r·min-1during middle and late log phases(48–72 h)to get moderate oxygen supply and avoid cell injury by vigorous stirring[2,6];in stationary phase(72–168 h),the stirring speed was 400 r·min-1to meet the basic demand of oxygen.No antifoams,acid or base was added in the batch fermentation process.

2.3.Dynamic monitoring of fermentation parameters

Cell density,FK520 concentration,and residual sugar were measured as reported[13].The average production rate of FK520 in 6 h is calculated by

where C1and C2are the concentration at the beginning and the end of one period,respectively.

DO and pH values were reflected by the detector instantly.Protein concentration in fermentation broth was quantified through Bradford method.Phosphorus was determined according to Fiske and Subbarow[14].Foaming was measured as follows:20 ml fresh fermentation broth was added to a 100 ml tube,then aerated at the rate of 50 ml·min-1and stirred with a glass rod for 1 min.The height of foam was measured to evaluate the foaming ability of the fermentation broth.

2.4.HPLC analysis of organic acids and permeability of cell membrane

HPLC method was used to find the changes of organic acids in the fermentation broth.The organic acids were extracted as follows:3 ml fermentation broth was mixed with 3 ml 6 mol·L-1HCl,reacted in 80°C water for 20 min,then cooled to room temperature;the supernatant was filtrated through 0.22 μm membrane,follows by a centrifugation at 10000×g for 10 min.The organic acids were determined as reported[15].

To determine whether acetate accumulation directly relates to membrane permeability changes during S.hygroscopicus fermentation,the extracellular and total FK520 were detected separately.The membrane permeability was measured from 24 h to 168 h according to[13].

2.5.Strategies to improve bioreactor culture conditions

A preliminary fed-batch fermentation mode was established according to the analysis of fermentation parameters in fermentation broth for S.hygroscopicus FS-35 batch fermentation in the bioreactor.The carbon source in fermentation medium was changed to 20 g·L-1dextrin.100 g·L-1glycerol was fed at the rate of 0.1 ml·min-1from 48 h to 144 h.A mixture of 30%oxygen and 70%air was used to supply oxygen from 48 h to 96 h.The stirring and aeration were maintained constant.

In this study,DO and pH values were instant values automatically detected and recorded.Each of the rest experiments was repeated three times.The data shown in the result are their mean value and error bars reflect the standard deviation.

3.Results and Discussion

To describe the process more accurately,the fermentation process was divided into four phases(lag phase,0–24 h;log phase,24–72 h;stationary phase,72–166 h;and decline phase,168 h later)in this study.

3.1.Relations between fermentation parameters and foaming

To investigate the influences of fermentation parameters on foaming,dynamic changes of cell density,FK520,foam height,pH value,DO value,extracellular protein concentration,residual sugar,and phosphorous of this fermentation process are shown in Fig.1.In 0–84 h,foam height[Fig.1(a)]decreased continuously and reached the lowest point at 84 h,so the increased foaming in stationary phase was not caused by components in initial medium;in 84–132 h,foam height increased significantly from 0.62 cm to 5.07 cm;in 132–168 h,it was basically unchanged.As foaming increased,cell density and phosphorus were stable while extracellular protein concentration and residual sugar decreased dramatically,suggesting that cell lysis and phosphorus are not the main reason for foaming,and proteins and carbohydrates are not foaming agents here.

In present study,to control oxygen supply efficiently,the stirring speed was first connected to DO value from 0 h to 72 h,with the lowest and highest stirring speeds at 200 and 500 r·min-1,respectively;then the stirring speed was set to 400 r·min-1from 72 h to 168 h to meet the oxygen demand and avoid cell injury.The actual DO value was close to the setting value from 0 h to 48 h,so oxygen supply was sufficient.However,the DO value dropped quickly and less than 10%(the setting value)for more than 12 h(60–72 h),during this period the pH value was 5.08(60 h),4.81(66 h),4.72(72 h)and 4.77(78 h)[Fig.1(c)].From the changes of DO and pH,it was noted that the deficiency of oxygen occurred first,then the pH value decreased continuously and maintained at low level for more than 18 h.In this situation,S.hygroscopicus FS-35 cells got insufficient oxygen and consumed glucose to produce organic acids such as lactate and acetate[16],which may cause pH dropping in the fermentation broth.Interestingly,foaming occurred and increased[Fig.1(a)]in early stationary phase(72–96 h)while oxygen was insufficient and the pH value was low.Therefore,the deficiency of oxygen and lower pH value may increase foaming.

3.2.Relations between acetate accumulation,membrane permeability and foaming

pH value is basically determined by medium components and their changes in the fermentation broth.Among them,organic acids produced by intracellular metabolism are important to decrease pH[17,18].As shown in Fig.2,lactate concentration was relatively low in the whole fermentation process while acetate concentration increased from 54.6 mg·L-1(48 h)to 320 mg·L-1(96 h)and maintained stable from 96 h to 168 h,suggesting that higher acetate production may be the most important reason to decrease pH.Thus oxygen deficiency may be the reason for acetate accumulation and decrease in pH value,consistent with previous study[19,20].

Fig.2.HPLC analysis of organic acids acetate and lactate in fermentation broth.

Acetate is lipophilic small molecule suppressing intracellular metabolism activity and cell growth,causing acidification of intracellular environment as well as cell membrane damages[12,21,22].In this study,to determine whether cell membrane damages occurred at high acetate concentration,the permeability of cell membrane was detected every 24 h(Fig.3).It was stable from 24 h to 96 h.However,from 96 h to 120 h,as acetate accumulated,the ratio of extracellular FK520 concentration to total FK520 concentration changed from 39%to 66%,increased by 69.2%.Therefore,although cell density was stable during the stationary phase,the membrane permeability increased dramatically.The improved permeability of cell membrane facilitates the efflux of intracellular metabolites[8],making the fermentation broth more complex,which might be the reason for increased foaming in present study.

Fig.3.Changes of extracellular FK520 concentration and membrane permeability.Experimental condition:with fermentation broth centrifuged at 6000 r·min-1 for 5 min to remove cells and other insoluble components,the supernatant mixed with ethanol at ratio of 1:1(volume ratio),and total FK520 being 0 mg·L-1 at 0 h.

Owning to stable operations(28 °C,400 r·min-1,1.5 vvm)during stationary phase of FK520 batch fermentation,it is reasonable to conclude that acetate accumulation caused by short period oxygen de ficiency is the reason for increased foaming.

3.3.Relations between foaming,FK520 production and acetate accumulation

FK520 production is affected by foaming[Fig.1(a)].In the early stationary phase(72–96 h),FK520 was synthesized at relatively high rate.The production rate of FK520 was 5.1 mg·L-1h-1at72 h,reached its maximum value 5.8 mg·L-1·h-1at 90 h and dropped to 5.2 mg·L-1·h-1at 96 h.Foaming began at 90 h.In the middle stationary phase(96–132 h),foam height reached its maximum at 132 h and the production rate of FK520 dropped significantly from 5.2 mg·L-1·h-1(96 h)to 2.2 mg·L-1·h-1(132 h).In the later time(132–168 h),foam height was relatively high and the production rate of FK520 dropped from 2.2 mg·L-1·h-1(132 h)to 1.0 mg·L-1·h-1(144 h)and 0.3 mg·L-1·h-1(168 h).These results suggest that foaming reduces not only the working volume of bioreactor but also FK520 production rate.

To investigate further the mechanism of foaming behavior and FK520 production,the foam height,FK520 production rate,and acetate concentration are analyzed together.As shown in Fig.A1,when acetate concentration reached relatively high level(320 mg·L-1)at 96 h,foaming increased and FK520 production decreased,indicating that more foaming caused by acetate accumulation is negative to FK520 production.On the one hand,acetate accumulation could lead to acidification of intracellular environment and suppress intracellular metabolism[23](see Fig.A2).On the other hand,acetate synthesis consumes lots of substances such as pyruvate and acetyl-CoA[16,24],directly or indirectly affecting FK520 production.This competition for substances makes the building blocks and precursors insufficient for FK520 synthesis,reducing its production rate.

3.4.Strategies to improve fermentation process

Acetate accumulation,which may decrease pH and increase foaming,occurred from 48 h to 96 h(Fig.2).Fermentation process optimization is a powerful approach to reduce carbohydrate consumption and acetate accumulation under aerobic conditions.A preliminary fed batch fermentation mode was established.First,20 g·L-1starch and 20 g·L-1dextrin in the fermentation medium were replaced by glycerol feeding in such a way that carbon concentration was controlled at a relatively low level;then,to meet the oxygen demand,a mixture of 30%oxygen and 70%air was used from 48 h to 96 h.DO and pH values and membrane permeability were determined separately,while the dynamic changes of cell density,foam height,acetate concentration and,FK520 production were also measured under the new fermentation mode.As shown in Fig.4(a),under this preliminary fed-batch fermentation mode,DO value maintained stable from 48 h to 168 h.The DO value above 20%means that the S.hygroscopicus FS-35 strain has sufficient oxygen in the broth for the metabolic.Thus the pH value is higher than that in Fig.1(c).From the changes of DO value,pH value and acetate concentration under fed-batch fermentation mode,we can conclude that as the oxygen limitation is eliminated,S.hygroscopicus FS-35 has sufficient oxygen for aerobic respiration,producing less acetate.Therefore,pH values are slightly higher than that in the batch fermentation mode.Interestingly,Fig.4(b)shows that the membrane permeability changed greatly under fed-batch fermentation mode compared to that under batch fermentation mode(Fig.3).This indicates that at lower acetate concentration,membrane permeability reduce dramatically.The data further suggest that acetate accumulation is a reason to increase membrane permeability,which may facilitate foaming.According to Fig.4(c),the maximum acetate concentration dropped from 320 mg·L-1to 157.4 mg·L-1,decreased by 50.8%;the maximum foam height was 3.74 cm at 168 h,reduced by 32.5%;and the maximum FK520 production was 421 mg·L-1,improved by 12%.The FK520 production was improved while acetate concentration and foaming of broth were effectively controlled under this preliminary fed-batch fermentation mode.These results further confirm our inference that acetate accumulation plays an important role in foaming and FK520 production.

Fig.4.Changes of fermentation parameters under fed-batch fermentation mode.(a)DO and pH;(b)extracellular FK520 and membrane permeability;(c)dry cell weight,FK520 production,foam height,and acetate concentration;experimental condition:with 30%pure oxygen and 70%air used instead of 100%air and 100 g glycerol·L-1 fed as carbon source.

4.Conclusions

The experimental results indicated that moderate acetate accumulation led to pH changes,increased membrane permeability,and finally more foaming.The synthesis of acetate that consumes substrates such as pyruvate might suppress FK520 production due to the sharing of substrates.To reduce acetate accumulation,improve FK520 production,and control foaming,a preliminary fed-batch fermentation mode was established,improving the production of FK520 by 12%and reducing foaming dramatically.The study shows the relations among intracellular metabolism,FK520 production and foaming more clearly and offers new insights into the FK520 fermentation.

Appendix

Fig.A1.HPLC results of organic acids in fermentation broth and cells.(a)Acetate and lactate;(b)fumaric acid and malic acid.

Fig.A2.Profiles of DO and pH in the whole fermentation process in the 7.5-L bioreactor.