The in?uences of pH control strategies on the distribution of 1,3-propanediols and 2,3-butanediols production by an isolated indigenous Klebsiella sp.
Ana-WS5
Hong-Wei Yen a ,?,Fang-Tzu Li a ,Jo-Shu Chang b ,c ,d
a
Department of Chemical and Materials Engineering,Tunghai University,Taichung 407,Taiwan b
Department of Chemical Engineering,National Cheng Kung University,Tainan 701,Taiwan c
Research Center for Energy Technology and Strategy,National Cheng Kung University,Tainan 701,Taiwan d
University Center for Bioscience and Biotechnology,National Cheng Kung University,Tainan 701,Taiwan
h i g h l i g h t s
Addition of 10g/L of lactic acid can create about a 30%increase in total diols production. The batch with the uncontrolled pH had the highest total diols production.
Both adding lactic acid and the pH ?uctuation strategy are simple and ef?cient methods.
a r t i c l e i n f o Article history:
Received 25January 2014
Received in revised form 21February 2014Accepted 25February 2014Available online 6March 2014Keywords:
pH adjustment Klebsiella
a b s t r a c t
In the simultaneous biological production of 1,3-propanediols (PDO)and 2,3-butanediols (BDO),glycerol was suggested to be a suitable carbon source.Extra addition of 10g/L of lactic acid can create about a 30%increase in total diols production the control batch.Several different pH control strategies were investigated.The results indicated that the batch with the uncontrolled pH had the highest total diols production among all pH control strategies,although it had the lowest productivity.Even the strat-egy of pH ?uctuation not enhance total diols production,it signi?cantly enhanced the productivity.The soluble metabolite products (SMPs)analysis also indicated that the pH ?uctuation will only affect BDO production,but had no impacts on the induction of more metabolites produced.Conclusively,both 1.Introduction
Both 2,3-butanediols (BDO)and 1,3-propanediols (PDO)are examples of important chemical building blocks which can be produced through a fermentation process.PDO is an important monomer that has been used to synthesize a new type of polyester,polytrimethylene terephthalate (PTT),as well as to carry out poly-condensation to produce polyesters,polyethers and polyurethanes (Zeng et al.,1991,2011).As well as PDO,microbial routes for BDO production have also been widely explored.BDO is a valuable chemical feedstock or ingredient that is widely used in foods,cosmetics,pharmaceuticals,plastics and the energy industry,with both chemical and biological production routes having been reported in previous studies (Ji et al.,2011;Syu,2001).In contrast to the related chemical processes,microbial BDO and PDO produc-tion has the advantages of low energy consumption and high selec-tivity.Therefore,many studies have focused on how to improve productivity as well as the yield through the microbial conversion of agricultural derivatives to BDO and PDO.
A large number of microbes have been reported to be able to convert hydrocarbons to BDO and PDO,including:Klebsiella sp.,Bacillus amyloliquefaciens,Paenibacillus polymyxa ,Serratia marces-cens ,Enterobacter aerugenes and Pseudomonas chlororaphis (Biswas et al.,2012;Jiang et al.,2012;Nakashimada et al.,2000;Rao et al.,2012).Among these,Klebsiella sp.seems to be the most promising species for industrial mass production of PDO and BDO (Cheng et al.,2007;Jun et al.,2009).During the fermentation process of diols,the pH level would rapidly drop to about 5.0,as observed in batch fermentation,in which the low pH level might have
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impacts on the growth of cells and related diols produc-tion.It has been shown that BDO synthesis is induced under acid supplementation,which suggests that BDO,as a neutral metabo-lite,counteracts a too high a level of acidi?cation(Syu,2001). Therefore,the addition of acid products is able to enhance BDO production by inducing enzymes involved in the BDO pathway (Celin′ska and Grajek,2009).With the addition of150mM of acetate into a batch culture at an initial pH of6.8,the production of BDO by Paenibacillus polymyxa reaches248mM,yielding 0.87mol per mol glucose consumed,where the ratio of acetate consumed to glucose consumed(A/C ratio)is calculated as 0.35mol acetate per mole of glucose(Nakashimada et al.,2000). It is clear that the various effects of pH and acetic acid on butanedi-ols fermentation are due to inhibition of growth by undissociated acetic acid and its subsequent effect on the energy(ATP)require-ment of cells(Zeng et al.,1990).Therefore,it has been suggested that the level at which pH is controlled is a detrimental factor for BDO production.To achieve a high BDO production rate by altering the pH level during cultivation,Petrov and Petrova proposed a forced pH?occulation method,which adjusted4pH(=1.0, 2.0 and3.0)at?xed time intervals.The results of their work showed that this pH control method could increase the?nal concentration of BDO by33%(Petrov and Petrova,2010).In our previous pub-lished work also revealed that pH control was an important deter-mined factor to the distribution of PDO and BDO(Yen et al.,2013).
The isolated Klebsiella strain used in this study is quite unique, which that it can produce both PDO and BDO simultaneously at a relatively high amounts.Although,many studies had revealed much higher titers of sole PDO/BDO obtained than that of this study.However,the effects of PDO and BDO distribution by the speci?c pH adjustment strategy and lactic acid adding for a strain capable producing PDO and BDO simultaneously have not been investigated.The results of this study can offer the insight on how to control the fermentation process toward BDO/PDO produc-tion.Several pH control strategies(pH adjusted to7.0per12/24h) and the addition of lactic acid are thus examined in this study to see they can enhance PDO and BDO production in an indigenous isolated Klebsiella strain.
2.Methods
2.1.Microorganism and culture conditions
Klebsiella sp.Ana-WS5was kindly
Chang from the National Cheng Kung
which a strain from the southern part of
The seed medium was grown on medium
erol,4g/L trypton and4g/L yeast extract.
ium consisted of70g/L glycerol(or glucose
KCl,1.38g/L NaH2PO4á2H2O,7.5g/L(NH4)2
0.26g/L MgSO4á7H2O,0.42g/L citric acid,8
and0.3mL/L of a trace element solution.
justed to8by a5N NaOH solution.The trace
sisted of34.2g/L ZnCl2, 2.7g/L FeCl3á6H2O,
0.3g/L H3BO3and23.8g/L CoCl2á6H2O(
2009).For the seed preparation,Klebsiella sp.
lated into50mL fresh seed medium in250
were incubated at150rpm and37°C for24
inoculated into the fermentation medium at
trials were performed at150rpm and37°
were conducted in a5L stirred-vessel
tion company,Taiwan)containing3L of
The pH was controlled by5N NaOH to a
respectively.For the trials of pH adjustment
matic pH control was performed to raise the the set times.All fermentation experiments were carried out at 37°C and100rpm with an aeration rate of1vvm.
2.2.Analytical methods
The optical density(OD)of the culture was assayed using a spectrophotometer(Genesys10UV,Thermo)at620nm with appropriate dilution.The value of the optical density was con-verted to dry cell weight(DCW)using a calibration equation.The concentrations of1,3-propanediols(PDO),2,3-butanediols(BDO), glycerol and SMPs(including succinic acid,acetic acid,ethanol and lactic acid)were analyzed with a high-performance liquid chromatography(HPLC)system(Agilent1100series),with an RI detector and column for organic acid analysis(ICSep ICE-Coregel 87H3column,Transgenomic).The temperature was set at 65°C,with0.008N H2SO4as the mobile phase at a?ow rate of 0.4mL/min(Cheng et al.,2010).
3.Results and discussion
3.1.Effects of carbon source and lactic acid addition on BDO and PDO production
Glycerol and glucose are known to be the suitable carbon sources for PDO and BDO production,respectively,due to the met-abolic pathways in which glycerol can be directly converted to PDO,while glucose can be converted to BDO by using phospho-enolpyruvate as the intermediate(Gungormusler et al.,2011; Jiayang et al.,2006).Nevertheless,the results of this study using glycerol and glucose as the carbon source for an isolated Klebsiella strain in a5L fermenter indicated that the batch with glycerol would have a higher BDO production than with glucose(Fig.1). As shown in Fig.1,glucose was obviously not a good carbon source for this strain to simultaneously produce PDO and BDO,due to the low concentrations of the resulting products.The production of BDO and PDO halted after about40h cultivation,and this because of the rapid fall in pH to about4.0,which occurred as the acids
Fig.1.BDO and PDO production by using glycerol and glucose as the carbon source respectively at70g/L in a5L fermenter with pH no control.
H.-W.Yen et al./Bioresource Technology159(2014)292–296293
In most cases,glycerol is assimilated
tions via two parallel metabolic pathways
ulon(catalyzed by glycerol dehydratase)for
and aerobic conditions were required for
through the glycolysis pathway(Metsoviti et
thus the primary products obtained in an
this study.The highest concentrations of
in this aerobic batch using glycerol were
respectively.For micro-aerobic conditions,
his colleagues obtained maximum PDO and
24.14and9.16g/L,respectively,in a
Klebsiella pneumoniae after a statistical
(Sattayasamitsathit et al.,2011).
Glycerol could be a suitable carbon
production as described previously,glucose
cell growth and BDO production before the
inhibits further activity,and thus a mixed
and glucose was examined.The results are shown in Fig.2,which indicates a mixed carbon source was not appropriate for BDO and
PDO production.The batch that contained only glycerol yielded the highest BDO and PDO concentrations,more than that produced by the other batches in which glucose was added to the medium. When adding glucose to the medium containing glycerol at70g/L, the cells seemed to?rst use glucose as the carbon source,leading to a fall in pH,which inhibited the digestion of glycerol and also re-duced the production of PDO.It was reported that a mutant strain of K.pneumoniae with deletion of the crr gene could use molasses as a co-substrate of glycerol to have the maximum PDO of81.2g/L produced which was60.4%greater than that obtained when glyc-erol was used alone(Oh et al.,2013).However,for this wild strain of Klebsiella,the mixture of glycerol and glucose didn’t enhance diols production.Therefore,the use of glycerol as the sole carbon source is suggested for BDO and PDO production.
To further con?rm the effects of the metabolic route on the pro-duction of BDO and PDO,varied amounts of lactic acid were added to the https://www.doczj.com/doc/d42906420.html,ctic acid and acetic acid are both known to be important metabolic products existing in the branching metabolic pathway of glucose converted to BDO.It had been reported that adding acetic acid can enhance BDO production by replacing the route of pyruvate to acetyl-CoA(Nakashimada et al.,2000;Zeng et al.,1990).It was thus supposed that adding small amounts of lactic acid might prevent the metabolic pathway from being di-as compared to28.2g/L in the control batch(without the addition of lactic acid).It was also reported that the production of sole BDO by Klebsiella oxytoca was enhanced in the presence of low levels (8g/L)of added sodium lactate.With3g/L lactate and an initial glucose level of98g/L,butanediol concentration and productivity increased164%with98%utilization of glucose(Qureshi and Cheryan,1989).Even though the addition of10g/L of lactic acid could lead to a30%increase in the total diols production compared to the control batch,it is still not economically ef?cient to have the cost balance.Therefore,it is proposed that a more practical way to enhance total diols production is through the control of pH,a relatively simple process parameter.
3.2.The effects of pH?uctuating every12or24h on BDO and PDO production
The results reported in the previous section indicate that glyc-erol might be a suitable carbon source for both BDO and PDO pro-duction.However,the pH level is known to be a determining factor that affects the production of diols chemicals.Petrov and Petrova revealed the effects of pH?uctuating with regard to enhancing sole BDO production with the cultivation of K.pneumoniae(Petrov and Petrova,2010).The authors concluded that?uctuating a?xed pH interval(4pH=1.0)every12h would lead to the greatest BDO production.However,the in?uences of pH?uctuating on simulta-neous BDO and PDO production remain unknown,and thus were examined in the current study.In this study,pH?uctuation was carried out every12or24h by repeatedly adjusting the pH level back to7.0,which was different to the?xed interval of pH adjust-ment(4pH=1.0)adopted by Petrov and Petrova.The results of the pH adjustment at?xed intervals are shown in Figs.4(every12h) and5(every24h).As shown in Fig.4,if the pH was adjusted back to7.0every12h,PDO production stopped after about40h of cul-tivation,although the BDO production continued to the maximum concentration of14g/L.However,when the glycerol was totally consumed,BDO seemed to be re-utilized as the carbon source, which caused a drop in the BDO concentration and led to the rise in the pH level.It is also noteworthy that the pH level could not be reduced to the same level as the value in the previous batch be-fore the pH adjustment.Similar results are seen in Fig.5for the pH adjustment back to7.0every24h.In this experiment,the PDO pro-duction was higher than that of the batch with the pH adjusted every12h,achieving a maximum concentration of25g/L of PDO. However,the levels of BDO production in both pH adjusted batches were very similar,at around15g/L.The ratios of PDO/BDO in both batches with pH adjustments every12or24h were1.38and1.76, respectively,far higher than the0.56found in the batch without a
Fig. 2.The effects of mixed glycerol and glucose(adding different amount of glucose to70g/L of glycerol)on PDO and BDO production in the shaker trials.Fig.3.Effects of lactic acid addition(0,5,10,15and20g/L)on the distribution of BDO and PDO by using70g/L glycerol as the carbon source in the shaker trials.
294
pH control.This suggests that the pH adjustment strategy was
more bene?cial to the production of PDO rather than BDO.Further-more,the productivity of PDO in the batches with pH adjustments was greatly enhanced,up to0.34and0.42g/L h compared to 0.093g/L h in the batch without pH control.All these results sug-gest that the pH is an important control factor,which could affect the metabolic route for PDO or BDO production.
https://www.doczj.com/doc/d42906420.html,parison of different pH control strategies on BDO and PDO production
The results presented in the previous section showed that the pH is a determining factor with regard to PDO and BDO production. Therefore,a comparison test was made by using glycerol as the sole carbon source and with the pH being controlled at?xed levels (6.0or7.0),the pH in?uctuating modes(every12or24h),and the control batch(pH without control).The results of BDO and PDO production with different pH control strategies are shown in Fig.6.It can be seen that the batch without pH control produced the highest?nal BDO concentration,but the lowest?nal PDO con-centration.The slow production rate of PDO also re?ected the slow glycerol consumption rate.It is noteworthy that the batches with the pH controlled at?xed levels of6.0or7.0saw signi?cant falls in BDO production,but increases in PDO production as compared to the batch without pH control.This con?rms the importance of pH level with regard to BDO and PDO production,as described pre-viously.The strategy of pH adjustment to6.0or7.0every12or 24h could have better performance than that of the pH being con-trolled at?xed levels of6.0or7.0,an approach that was supported by the results of Petrov and Petrova(2010).The highest of all batches examined here was30g/L of BDO in the batch without pH control after a long period of about200h cultivation.
A comparison of several kinetic parameters obtained for all the pH control strategies is shown in Table1,which indicates that pH adjustment at a?xed level or adjusted at?xed time intervals can obtain a higher ratio of PDO/BDO than that of a batch without pH control.The ratios of PDO/BDO are5.35and4.05in batches with pH controlled at7.0and6.0respectively as compared the val-ues of1.38and1.76of pH adjustment per12and24h.The low val-ues of PDO/BDO suggest that the production of BDO is signi?cantly inhibited,while the pH is controlled at a?xed level of6.0and7.0. The analysis of soluble metabolite products(SMPs)among all batches is shown in Table2,which indicates the batches with a higher BDO production also leading to higher SMPs observed.Most SMPs(acetic acid,lactic acid,succinic acid and ethanol)measured in the broth were located on the diverted routes to BDO produc-tion.That implied the more mass?ux diverted to the route of BDO production potentially being able to produce more SMPs. Since the?uctuation of pH could enhance more BDO production, it would certainly increase SMPs concentration as compared to the batches with pH uncontrolled.Nevertheless,no matter what kind of pH strategy was applied,the ratio of total diols/SMPs didn’t have signi?cant difference,which indicated the total diols produc-tion played a major role in the metabolism of this indigenous strain-Klebsiella sp.Ana-WS5.Therefore,this strain should have the potential being the producer of BDO and PDO,due to its high value of total diols/SMPs.
Fig.4.The fermentation pro?les in the batch of pH adjusted to7.0per12h with
70g/L glycerol.
Fig.5.The fermentation pro?les in the batch of pH adjusted to7.0per24h with
70g/L glycerol. https://www.doczj.com/doc/d42906420.html,parison of BDO and PDO production among all batches of different
control strategies.
4.Conclusion
Sole glycerol is suggested as a suitable carbon source for simul-taneous BDO and PDO production in the cultivation of Klebsiella sp. Ana-ws5.Extra adding lactic acid can enhance total diols produc-tion.Although the strategy adjusting the pH to7.0every12or 24h did not enhance the maximum BDO and PDO concentration, it did greatly improve productivity compared to the batch without a pH control.In conclusion,both lactic acid addition and the adjusting of pH level at?xed time intervals(12/24h)are simple and ef?cient ways to enhance simultaneous BDO and PDO production.
Acknowledgements
The authors gratefully acknowledge the?nancial support this study received from Taiwan’s National Science Council under Grant No.102-2623-E-029-001-ET.
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Table1
Comparison of PDO/BDO,maximum PDO and BDO productivity among all batches.
PDO/BDO Maximum PDO(g/L)Maximum BDO(g/L)Total diols(g/L)PDO productivity(g/L h)BDO productivity(g/L h)
Control*0.561730.347.30.10.13
pH7 5.3518.2321.20.730.14
pH6 4.0521.5 5.326.80.660.2
pH7per12h 1.3819.113.832.90.340.15
pH7per24h 1.7625.314.439.70.420.18
*The batch without pH control.
Table2
Soluble metabolite products(SMPs)measurement under different pH conditions.
Succinic acid(g/L)Lactic acid(g/L)Acetic acid(g/L)Ethanol(g/L)Total diols(g/L)SMPs+(g/L)Total dios/SMPs
Control*0.30.80.1 2.347.350.80.93 pH70.60.80.3 1.621.224.50.87 pH60.7 1.00.4 1.726.830.60.88 pH7per12h0.7 1.20.3 2.532.937.60.88 pH7per24h0.7 1.30.4 3.139.745.20.88
*The batch without pH control.
+Sum of succinic acid,lactic acid,acetic acid,ethanol and total diols.
296H.-W.Yen et al./Bioresource Technology159(2014)292–296