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JOURNALOFBIOSCIENCEANDBIOENGINEERING

Vol. 98, No. 4, 263268. 2004

High Butanol Production by Clostridium saccharoperbutylacetonicum

N1-4 in Fed-Batch Culture with pH-Stat ContinuousButyric Acid and Glucose Feeding Method

YUKIHIRO TASHIRO,1 KATSUHISA TAKEDA,1 GENTA KOBAYASHI,1*KENJI SONOMOTO,1 AYAAKI ISHIZAKI,1 ANDSADAZO YOSHINO2

Laboratory of Microbial Technology, Department of Bioscience and Biotechnology, Faculty of Agriculture,Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan1 and Laboratory of Applied

Microbiology, Department of Bioscience and Biotechnology, Faculty of Agriculture,Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan2

Received 21 April 2004/Accepted 9 July 2004

A pH-stat fed-batch culture by feeding butyric acid and glucose has been studied in an acetone

butanolethanol (ABE) fermentation using Clostridium saccharoperbutylacetonicum N1-4. Thespecific butanol production rate increased from 0.10 g-butanol/g-cells/h with no feeding of butyricacid to 0.42 g-butanol/g-cells/h with 5.0 g/lbutyric acid. The pH value in broth decreases with bu-tyric acid production during acidogenesis, and then butyric acid reutilization and butanol pro-duction result in a pH increase during solventogensis. The pH-stat fed-batch culture was per-formed to maintain a constant pH and butyric acid concentration in the culture broth, but feedingonly butyric acid could not support butyric acid utilization and butanol production. Subsequent-ly, when a mixture of butyric acid and glucose was fed, butyric acid was utilized and butanol wasproduced. To investigate the effect of the feeding ratio of butyric acid to glucose (B/G ratio), sev-eral B/G ratio solutions were fed. The maximum butanol production was 16 g/land the residualglucose concentration in broth was very low at a B/G ratio of 1.4. Moreover, yields of butanol inrelation to cell mass and glucose utilization were 54% and 72% higher in pH-stat fed-batch cul-ture with butyric acid than that of conventional batch culture, respectively.

[Keywords: acetonebutanolethanol fermentation, pH-stat fed-batch culture, high butanol production, B/G ratio,Clostridium saccharoperbutylacetonicumN1-4]

Acetonebutanolethanol (ABE) fermentation was widelycarried out industrially during the first half of last century(1), but later it could not compete economically with petro-chemical synthesis. Currently using renewable resources,this fermentation is becoming very attractive for the produc-tion of chemicals and liquid fuels (2, 3). At present, consid-erable research has been conducted on the type of ABE fer-mentation system (4, 5), including batch culture (6, 7) orfed-batch culture (8, 9) integrated with a butanol removal

process, and continuous culture with concentrated cell mass(10, 11) or immobilized cell mass (12, 13). In previous stud-ies, the yields of butanol to glucose were under 30%, andthe residual glucose concentrations in broth were very high.To date, a highly efficient butanol production system hasnot yet been established.

ABE-producing clostridia possess two distinct character-istic phases in energy acquiring pathway, specifically acido-genesis and solventogenesis (1, 14). Typically, during acido-genesis, cell growth is exponential and products are aceticacid and butyric acid with ATP formation. Accumulation ofthese organic acids results in a decrease in the pH of the

broth. During solventogenesis, cell growth enters the station-ary phase and the above organic acids are reutilized and ace-tone, butanol and ethanol are produced. This reutilization oforganic acids results in a pH increase of the broth. It is re-ported that organic acid production is enhanced at higherpH, while solvents are mainly produced at lower pH (1518). On the other hand, since the addition of organic acids tothe growth medium has been shown to stimulate solventproduction and protect against the degeneration of ABE-

producing clostridia, it is suggested that organic acids inbroth trigger a metabolic shift from acidogenesis to solvento-gensis, although the exact mechanism is still unknown (15,1922). Thus, we noted that butanol could be producedeffectively at lower pH by feeding organic acids such asacetic acid or butyric acid. Presently, there is no report onthis feeding method in ABE fermentation.

The aim of this study was to establish a high butanol pro-duction system using a high butanol producer, Clostridiumsaccharoperbutylacetonicum N1-4. Here, we investigatedthe effect of organic acids on solvent production and appliedthe pH-stat continuous substrate feeding method to maintaina lower pH and butyric acid concentration. As a result, thebutyric acid concentration and pH value of the broth could

be maintained by feeding butyric acid and glucose, and bu-* Corresponding author. e-mail: gentak@agr.kyushu-u.ac.jpphone/fax: +81-(0)92-642-3021

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tanol production reached a level similar to that of conven-tional batch culture. Furthermore, this system enabled theglucose concentration in the broth to be controlled at a verylow value, approximately 0, and yields of butanol in relationto cell mass and glucose in the pH-stat fed-batch culture in-

creased 54% and 72%, respectively.

MATERIALS AND METHODS

Bacterial strain C. saccharoperbutylacetonicum N1-4ATCC 13564 was used in this study (7). The culture was kept asspores in fresh potato glucose (PG) medium at 4C. One ml ofspore suspension was transferred aseptically to 9 ml of PG mediumand heat-shocked in boiling water for 1 min and cultured at 30Cfor 24 h (2) and used as an inoculum.

Media Triptoneyeast extractacetate (TYA) medium wasused for the pre-culture and main culture, and contained the fol-lowing compounds per liter of distilled water (7): 1050 g glu-cose, 2 g yeast extract, 6 g tryptone, 3 g CH

3COONH

4, 0.3 g

MgSO47H2O, 0.5 g KH2PO4, and 10 mg FeSO4

7H2O. Various

amounts of butyric acid and sodium acetate were added to the me-dium separately in some experiments. In the case of acetate addi-tion, 0.77 g of ammonium sulphate were used instead of 3 g ofCH

3COONH

4. In all experiments, the initial pH of the medium was

adjusted to 6.5 with 1 M NaOH or 1 M HCl and the medium wassterilized at 115C for 15 min.

Culture conditionsAcid addition A batch culture was carried out statically at

30C in a 500-ml Erlenmeyer flask with a 300-ml working volumethat included a 10% inoculum volume. Following inoculation, thebroth was sparged with filtered oxygen-free nitrogen gas to main-tain strict anaerobic conditions. Periodically, samples were with-drawn.

pH-statfed-batchculture A pH-stat fed-batch culture wascarried out in a 1-ljar fermentor with a 400-ml working volume.The initial glucose concentration was 10 g/l in TYA medium andapproximately 60 g/l or 180 g/l butyric acid and 180 g/l glucosesolution were prepared separately as feeding solutions. The pH ofthe broth was monitored using a pH controller (PHC-2201; Able,Tokyo). The pH-stat fed-batch culture was started at the pH transi-tion point, which indicated the shift to solventogenesis. The feed-ing ratio of butyric acid to glucose (B/G ratio) was set at 0.42,0.80, 1.0, 1.15, 1.4 and 1.6 using the concentration controller(Gradicon III, AC-5900 type; ATTO, Tokyo). Unless otherwisestated, the culture conditions were the same as described above.

Analyticalmethods The cell concentration was estimatedby optical density (OD) with a spectrophotometer (V-530; JASCO,Tokyo) and the dry cell weight (DCW) was calculated using a pre-determined correlation between OD at 562 nm and DCW. Acid and

solvent concentrations in the supernatant were determined with agas chromatograph (6890A; Agilent Technologies, Palo Alto, CA,USA) equipped with a flame ionization detector and a 15 m capil-lary column (Innowax; i.d. 0.53 mm; 19095N-121; Agilent Tech-nologies). The oventemperature was programmed to increase from50C to 170C at the rate of 10C/min. The injector and detectortemperatures were set at 250C. Helium was the carrier gas andwas set at a flow rate of 3.7 ml/min. Iso-butanol was used as theinternal standard with 1 M HCl. The glucose concentration in thesupernatant was determined with a glucose analyzer (BF-4; OjiScientific Instrument, Osaka).

Calculations The following equation was used to calculatethe specific growth rate.

Specific growth rate (h1) ln (X2/X

1)/ (t

2 t

1)

where Xis the cell concentration (g/l) and t is the sampling time

(h).The following equation was used to calculate the specific pro-

duction rate.

Specific production rate (g/g/h) (C2C

1)/ (t

2 t

1)/X*

where Cis the concentration of product (g/l), tis the sampling time(h) andX* is the mean cell concentration of t1and t

2(g/l).

The following equations were used to calculate butanol yields.

Butanol yield to cell mass (g-butanol/g-cells) P/Xmax

wherePis the butanol production (g/l) and Xmax

is the maximumcell concentration (g/l).

Butanol yield to glucose utilization (g-butanol /g-glucose)P/S

wherePis the butanol production (g/l) and Sis the glucose utiliza-tion (g/l).

RESULTS

Effect of organic acid addition on solvent productionTo investigate the effect of added acetate on solvent produc-tion by C. saccharoperbutylacetonicumN1-4, batch cultureswere carried out in TYA medium containing 0, 2.1, 4.2, and6.2 g/lacetate. During the initial culture with C. saccharo-perbutylacetonicumN1-4, the specific growth rates and spe-cific butanol production rates in the presence of acetatewere similar to those in the absence of acetate (Table 1). Inthe initial exponential phase, however, the specific acetoneproduction rates were 0.020, 0.046, 0.073 and 0.088 g/g/h inTYA medium containing 0, 2.1, 4.2, and 6.2 g/lacetate, re-spectively. These results indicated that the addition of ace-tate to the growth medium could not significantly enhance

butanol production or cell growth but acetone productionwas enhanced.To examine the effect of added butyric acid, the batch

cultures were carried out in TYA medium containing 0, 1.5,3.2, and 5.0 g/l butyric acid (Table 2). Since the specificgrowth rates between 0 h and 3 h decreased with increasinginitial butyric acid concentration, it was suggested that cellgrowth was inhibited by butyric acid in broth. The amountsof butanol and acetone produced in TYA medium contain-ing 5.0 g/lbutyric acid were larger than those produced inTYA medium containing other butyric acid concentrations.Furthermore, during the initial acidogenesis phase of C. sac-charoperbutylacetonicumN1-4 culture, the specific butanolproduction rates were 0.25, 0.39 and 0.42 g/g/h in the pres-

ence of 1.5, 3.2 and 5.0 g/lbutyric acid, respectively, and0.10 g/g/h in the absence of butyric acid. In the case of5.0 g/lbutyric acid addition, in particular, the specific bu-tanol production rate was similar to that during solvento-

TABLE 1. Effect of acetic acid in ABE fermentation

Acetic acid(g/l)

Specificgrowth ratea

(h1)

Specific production rateb(g/g/h)

Butanol Acetone

0 0.22 0.10 0.0202.1 0.24 0.098 0.0464.2 0.22 0.088 0.0736.2 0.21 0.073 0.088

a Specific growth rate between 0 h and 5h.b Specific production rate between 0 h and 5h.

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genesis in batch culture (data not shown). Surprisingly, theethanol production in the presence of butyric acid was lowerthan that in its absence. These data indicated that butyricacid in broth not only promoted butanol and acetone pro-duction but also induced butanol production during the ini-tial culture of C. saccharoperbutylacetonicumN1-4. Fromthe above results, we decided to use butyric acid as a feed-

ing solution for the pH-stat fed-batch culture in ABE fer-mentation.

pH-stat fed-batch culture with butyric acid and glucoseTo obtain butanol from butyric acid in pH-stat fed-batchcultures, the initial glucose concentration was decreasedfrom 50 to 10 g/lin TYA medium. Butyric acid solution wasfed to maintain a constant pH at the transition point in brothafter 79 h when acidogenesis shifted to solventogenesis.Figure 1 shows profiles of pH, butanol, butyric acid andglucose concentrations with feeding only butyric acid andwithout feeding it. The pH of the broth could be maintainedat pH 5.5 during feeding of only butyric acid, while the pHincreased to 6.9 without feeding. The cell growth was not

inhibited with feeding of only butyric acid (data not shown).However, the highest butanol production was 3.1 g/l withfeeding only butyric acid, while 3.0 g/l butanol was pro-

duced without feeding butyric acid. In addition, the glucosein the broth was utilized completely within 12 h and butyricacid utilization and butanol production were not observed ineither culture after 12 h of cultivation. It was suggested thatthe bioconversion from butyric acid to butanol by C. sac-charoperbutylacetonicum N1-4 required the energy obtainedby glycolysis from glucose.

Subsequently, to investigate the necessity of an energysource for butyric acid utilization and butanol production,mixed solutions of 60 g/lbutyric acid and 150 g/lor 250 g/lglucose were fed separately using the same method. The pHof the broth could be maintained at pH 5.45.5 during thefeeding of butyric acid solution containing 150 g/lor 250 g/lglucose. As shown in Fig. 2, butyric acid utilization and bu-tanol production were confirmed following feeding of thebutyric acid solution containing glucose. Although the re-sidual glucose concentration in the broth using the feedingsolution containing 250 g/lglucose was higher than that con-taining 150 g/lglucose, the profiles of butyric acid utiliza-tion and butanol production were very similar. These data

indicated that butyric acid utilization and butanol produc-tion were not affected by the residual glucose in pH-stat fed-batch culture with butyric acid. Therefore, it was suggested

TABLE 2. Effect of butyric acid in ABE fermentation

Butyricacid(g/l)

Production Specificgrowth ratea

(h1)

Specific butanolproduction rateb

(g/g/h)

Solventyieldc

(g/g)Acetone (g/l) Butanol (g/ l) Ethanol (g/ l)

0 3.6 14 1.6 0.28 0.10 0.43

1.5 4.3 16 1.4 0.26 0.25 0.443.2 5.1 16 0.99 0.22 0.39 0.465.0 6.0 17 1.1 0.21 0.42 0.49

a Specific growth rate between 0 h and 3h.b Specific butanol production rate between 0 h and 3 h.c Total solvent production per glucose utilization.

FIG. 1. Time course of pH-stat fed-batch culture with feeding bu-tyric acid and batch culture. Symbols: circles, butanol concentration inbroth; triangles, butyric acid concentration in broth; squares, glucoseconcentration in broth; rhombuses, pH of broth; closed symbols, pH-stat fed-batch culture; open symbols, batch culture. The dashed line in-dicates the onset of pH-stat fed-batch culture.

FIG. 2. Time course of pH-stat fed-batch culture with feeding asolution of butyric acid and glucose. Symbols: circles, butanol concen-tration in broth; triangles, butyric acid concentration in broth; squares,glucose concentration in broth; closed symbols, 250 g/l glucose inmixed solution; open symbols, 150 g/lglucose in mixed solution. Thedashed line indicates the onset of pH-stat fed-batch culture.

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that an optimum ratio of butyric acid to glucose (B/G ratio)in the feeding solution could be used to control the residualglucose concentration to zero in the pH-stat fed-batch cul-ture with butyric acid.

Effect of B/G ratio on butanol production and residual

glucose concentration To examine the effect of the re-lationship between the butyric acid and glucose concentra-tions in pH-stat fed-batch culture with butyric acid, the con-centration controller was used to control the B/G ratio. B/Gratios were set at 0.42, 0.80, 1.0, 1.15, 1.4 and 1.6. The pHof the broth could be maintained at a constant value duringthe feeding of butyric acid and glucose, although the pHtransition points were different in each culture in the rangeof 5.15.4. The profiles of butanol, butyric acid and residualglucose concentrations in the broth are shown in Fig. 3 atvarious B/G ratios. In each experiment, the butyric acid con-centration was controlled at a similar level during the feed-ing of butyric acid and butanol production reached morethan 13g/l, comparable to approximately 14 g/lin conven-

tional batch culture. In the case of the B/G ratio of 1.4, the

maximum butanol production of 16 g/lwas obtained after36 h of cultivation (Fig. 3e). On the other hand, the residualglucose concentrations in the broth were different for thecultures at various B/G ratios. At B/G ratios of 0.42 and0.80, residual glucose in the broth increased gradually and

reached 18 g/l(Fig. 3a) and 13 g/l (Fig. 3b), respectively,while the residual glucose concentrations in broth were main-tained at significantly low levels at B/G ratios of more than1.15 (Fig. 3df). At a B/G ratio of 1.6, in particular, residualglucose was not observed in the broth after 15 h, assumingthat the glucose utilization rate was 100% (Fig. 3f). Conse-quently, butanol could be produced at a low glucose concen-tration (01.7 g/l) in the presence of butyric acid in this sys-tem (Fig. 3df).

Comparison of solvent production between pH-stat fed-batch culture with butyric acid and conventional batchculture Solvent production and butanol yield in relationto cell mass and glucose utilization in pH-stat fed-batch cul-tures with butyric acid at various B/G ratios and conven-

tional batch culture have been summarized in Table 3. The

FIG. 3. Time course of pH-stat fed-batch cultures at various B/G ratios: (a) 0.42; (b) 0.80; (c) 1.0; (d) 1.15; (e) 1.4; (f) 1.6. Symbols: circles,butanol concentration in broth; triangles, butyric acid concentration in broth; squares, glucose concentration in broth. The dashed lines indicate theonset of pH-stat fed-batch culture.

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maximum butanol production in pH-stat fed-batch culturewas similar to that in batch culture and the produced butanolinhibited cell growth and butanol production (23). Interest-

ingly, ethanol was not produced in any of the pH-stat fed-batch cultures with butyric acid, while approximately 1.4 g/lethanol was generally produced in conventional batch cul-ture. Furthermore, the yields of butanol in relation to cellmass and glucose utilization were higher in pH-stat fed-batch culture than in batch culture. In particular, the maxi-mum yields, 3.7 and 0.55 in relation to cell mass and glu-cose utilization, respectively, were obtained in pH-stat fed-batch culture at a B/G of 1.4 and these yields increased 54%and 72% from that of batch culture. From these data, it wassuggested that the metabolic flux of butyric acid utilizationto butanol production was enhanced and that the metabolicflux to ethanol production was repressed. In conclusion, weestablished a high yielding butanol production system with

butyric acid and glucose in pH-stat fed-batch culture.

DISCUSSION

pH-stat fed-batch cultures have been applied for polyhy-droxyalkanoate or recombinant protein production (24, 25).The pH-stat fed-batch cultures facilitate maintaining the sub-strate concentration at a low level in the broth or repressingundesirable by-products caused by substrate at a high level.To the best of our knowledge, there is no report on the appli-cation of pH-stat fed-batch culture in ABE fermentation. Inthis paper, we first reported on an improved butanol produc-tion system in fed-batch culture with a pH-stat continuous

butyric acid and glucose feeding method.Butyric acid in broth, particularly undissociated butyricacid, has been shown to trigger solvent production by C.acetobutylicum(17, 2628). Moreover, increases in yieldand production of solvents have been reported following theaddition of acetic acid and butyric acid to cultures of C.acetobutylicumand C. beijerinckii(15, 1922). It was alsoobserved with C. saccharoperbutylacetonicumN1-4, that bu-tyric acid elevated the specific butanol production rate, ace-tonebutanol production and yield of solvent with increas-ing the added butyric acid concentration (Table 2), althoughacetic acid only could enhance acetone production (Table 1).On the other hand, it is well known in ABE fermentationthat the pH of the broth affects the organic acid or solvent

production (1518). At high pH, organic acids are mainly

produced, while at low pH, solvent production is stimulated.Solvent production by C. saccharoperbutylacetonicumN1-4was also shown to be enhanced at pH 5.0, similar to otherABE-producing clostridia (data not shown).

When only butyric acid was fed, butanol production was

similar to that without feeding butyric acid after glucose de-pletion (Fig. 1). However, when a solution of butyric acidcontaining glucose was fed, butyric acid utilization and bu-tanol production were observed (Fig. 2). Previous research-ers reported that butyric acid was reutilized and convertedto butanol via three metabolic enzymes, i.e., acetoacetyl-CoA: acetate/butyrate: CoA transferase, NADH-dependentbutyraldehyde dehydrogenase and NADH-dependent butanoldehydrogenase (1, 19). Since these dehydrogenases requirereducing power, such as NADH obtained by glycolysis fromglucose, no butanol production by feeding of only butyricacid may result from insufficient NADH due to the absenceof glucose. Consequently, energy sources like glucose are

necessary for butyric acid utilization and butanol produc-tion.In pH-stat fed-batch cultures with butyric acid, the B/G

ratio affected the residual glucose concentration in broth,but not the butyric acid or butanol profiles (Fig. 3). Al-though the residual glucose concentration in broth increasedgradually at B/G ratios less than 1.0 (Fig. 3ac), the residualglucose concentration in broth was controlled at zero with aB/G ratio of 1.6 (Fig. 3f). In other words, glucose fed withbutyric acid was immediately utilized in this culture, indi-cating that the glucose utilization rate was constantly 100%.Since it was previously reported that no feeding methodcould control the residual glucose concentration in broth atzero constantly, we are the first to achieve a glucose utiliza-

tion rate of 100% in an ABE fermentation. As expected,higher yields of butanol in this culture were obtained, atapproximately 1.5-fold those in conventional batch culture(Table 3). Furthermore, specific butanol production ratesabove 0.10 g/g/h were maintained until 39 h of cultivation ata B/G ratio of 1.6 (Fig. 3f), while specific butanol produc-tion rates in conventional batch culture rapidly decreasedduring the late exponential phase and finally reached zerowithin 30 h (data not shown). It was suggested that theseimprovements were attributed to the synergistic effect oflower pH control (

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