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Enzyme and Microbial Technology 41 (2007) 149–155

Direct l-lactic acid fermentation with sago starch by a novelamylolytic lactic acid bacterium, Enterococcus faecium

Keisuke Shibata a, Dulce M. Flores b, Genta Kobayashi a, Kenji Sonomoto a,c,∗a Laboratory of Microbial Technology, Division of Microbial Science and Technology, Department of Bioscience and Biotechnology,

Faculty of Agriculture, Graduate School, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japanb Department of Food Science and Chemistry, University of the Philippines Mindanao, Bago Oshiro, Tugbok 8000, Philippines

c Laboratory of Functional Food Design, Department of Functional Metabolic Design, Bio-Architecture Center,Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan

Received 15 September 2006; received in revised form 15 December 2006; accepted 16 December 2006

bstract

Novel amylolytic lactic acid bacterium from puto, fermented raw rice in Philippine, was isolated and characterized. The strain was identifieds Enterococcus faecium No. 78 (BIOTECH 10375) by sugar fermentative test and 16S rDNA sequence analysis. Optimum pH and temperatureere 6.5 and 30 ◦C, respectively. Direct l-lactic acid fermentation was carried out with various starches, lactic acid productivity with sago starcheing similar to that with glucose. Yield of lactic acid from sago starch was higher than those from glucose and other starches. Strain No. 78as superior to the other amylolytic lactic acid bacteria so far reported on the direct lactic acid fermentation with starches and produced lactic

cid of high optical purity (98.6%). In direct lactic acid fermentation with starch, continuous culture has hardly been reported. Continuous cultureystem with high cell density of strain No. 78 showed higher lactic acid productivity (3.04 g l−1 h−1) than those of batch culture (1.105 g l−1 h−1)nd conventional continuous culture (1.56 g l−1 h−1). Even if the dilution rate was increased up to 0.26 h−1, the residual starch concentration wasontrolled at moderately low level below 3.24 g l−1.

2007 Elsevier Inc. All rights reserved.

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eywords: Enterococcus faecium; Direct l-lactic acid fermentation; Sago starc

. Introduction

Over the last decade, lactic acid production has attracted con-iderable attention [1,2], because lactic acid is a raw material forynthesis of polylactic acid which is an essential biodegradablelastic material [3]. Presently, of the 80,000 ton of lactic acid pro-uced annually worldwide about 90% is produced by lactic acidermentation [4]. Fermentative production has advantage thatn optical pure product can be obtained by choosing a microbialtrain to produce only the isomers, whereas synthetic produc-

ion always results in a racemic mixture of lactic acid. It is alsoossible to use renewable resources as substrates, such as starchn fermentative production. Renewable resources do not give

∗ Corresponding author at: Laboratory of Microbial Technology, Division oficrobial Science and Technology, Department of Bioscience and Biotech-

ology, Faculty of Agriculture, Graduate School, Kyushu University, 6-10-1akozaki, Higashi-ku, Fukuoka 812-8581, Japan. Tel.: +81 92 642 3019;

ax: +81 92 642 3019.E-mail address: sonomoto@agr.kyushu-u.ac.jp (K. Sonomoto).

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141-0229/$ – see front matter © 2007 Elsevier Inc. All rights reserved.oi:10.1016/j.enzmictec.2006.12.020

ny net contribution of carbon dioxide to atmosphere, as do theimited oil- and fossil fuel-based sources.

Sago starch obtained from sago palm is a cheap and verynteresting substrate for lactic acid fermentation. Sago palm isigh energy crops widely cultivated in peat soil land in tropi-al area. Since sago palm can accumulate solar energy in theorm of starch by reducing carbon dioxide, much attention iseing denoted to this crop from the standpoint of the viewsor the countermeasure of the elevation of carbon dioxide inhe atmosphere to cause green house effect. Twenty-five tona−1 year−1 of starch productivity has been confirmed in sagolantation which is under development stage in Sarawak Statef Malaysia [5]. It is so far the highest record of productivitymong the starchy crops in the world [5]. We have establishedhe efficient lactic acid production using hydrolyzed sago starchy Lactococcus lactis IO-1 [6]. Before fermentation, enzymatic

retreatment of liquefaction and saccharification is essential,owever, in which starch is eventually hydrolyzed to glucoset 90 ◦C for 30 h. Simplification of the two sequential processess desired for an efficient lactic acid fermentation.

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Amylolytic lactic acid bacteria are able to simultaneouslyydrolyze and ferment starch to lactic acid. Recently, only aew amylolytic lactic acid bacteria have been isolated, such asactobacillus amylophilus JCM 1125 [7,8], L. amylovorus JCM126 [9], L. plantarum A6 [10] and L. manihotivorans LMG8010T [11]. Among these strains, only L. amylophilus JCM125 was reported as l-(+)-isomer producer, but its ability toroduce lactic acid seemed to be lower than those of the otherhree strains.

Recently, we isolated a novel lactic acid producer No. 78 fromuto in Philippine, which can be realized as an amylolytic one.he purpose of this study was to investigate the direct l-lacticcid fermentation with sago starch by employing strain No. 78ue to elimination of pretreatment process of starch. Thus far,he continuous fermentation has hardly been reported in directactic acid fermentation with starch. This study also describesontinuous lactic acid fermentation with sago starch without anyretreatment of starch.

. Materials and methods

.1. Identification of the isolate No. 78

Strain No. 78 was identified by 16S rDNA sequence and sugar fermen-ation pattern. Partial 16S rDNA region of strain No. 78, corresponding toositions 8-519 of Escherichia coli 16S rDNA, was analysed using a set of primerA and 519B. These primers possess the sequence as follows: 8A, 5′-AGA-

TTTGATC(C/A)TGGCTCA-3′; 519B, 5′-ATTACCGCCGCTGCTG-3′. The

otal genomic DNA of strain No. 78 was extracted from the cells treated withysozyme (Seikagaku, Tokyo, Japan) and labiase (Seikagaku), according to the

anufacture protocol, and used for PCR template. PCR was performed with TaqNA polymerase (Promega, Madison, WI, USA) under the condition consisting

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ig. 1. Schematic diagram of continuous lactic acid fermentation with cell-recyclingicrofiltration module, (4) agitator of 1-l jar bioreactor, (5) agitator of 5-l jar bioreacto

eservoir, (13) bleed out reservoir, (14) 5 M NaOH, (15) pH controller and (16) turbid

l Technology 41 (2007) 149–155

f denaturation at 94 ◦C for 30 s, annealing at 53 ◦C for 30 s and polymerizationt 72 ◦C for 1 min. The amplified products were purified by using QIAquickCR Purification Kit (Qiagen, Hilden, Germany). Then, it was sequenced withRISA-384 DNA sequencer (Shimadzu, Kyoto, Japan) using the primers shownbove.

Sugar fermentation pattern of strain No. 78 was tested by the API 50 CHLystem (bioMerieux, Marcy I’Etoile, France). The obtained pattern was com-ared with those of reference strains, described by Manero and Blanch [12].

.2. Microorganisms and growth medium

The microorganisms used were the isolated No. 78 and the previouslyeported amylolytic lactic acid bacteria, L. amylovorus JCM 1126 and L.anihotivorans JCM 12514 (LMG 18010T). Stock culture was maintained

t −80 ◦C in vial containing 30% glycerol until used. MRS medium wassed as a growth medium with the following components (l−1): 10 g pep-one (Difco, Detroit, MI, USA), 8 g beef extract (Nacalai Tesque, Kyoto,apan), 4 g yeast extract (Nacalai Tesque), 2 g K2HPO4 (Nacalai Tesque),g CH3COONa·3H2O (Nacalai Tesque), 2 g tri-ammonium citrate (Nacalaiesque), 0.2 g MgSO4·7H2O (Nacalai Tesque), 0.05 g MnSO4·4H2O (Nacalaiesque), 1 ml Tween 80 (Nacalai Tesque) and about 20 g soluble starch (Nacalaiesque) as carbon source.

.3. Carbon sources

Soluble, corn, wheat and potato starches, malotose and glucose were pur-hased from Nacalai Tesque. Sago starch was obtained through the Universityf Malaysia, Sarawak, (UNIMAS, Kuching, Sarawak, Malaysia), and stored atoom temperature until use. In this study, carbon sources were used at an initialoncentration of carbon source of about 20 g l−1.

.4. Cultivation system

Fermentation was carried out at an agitation rate of 200 rpm in a 1-l jarermentor set-up with a working volume of 400 ml. L. amylovorus JCM 1126

and bleeding. (1) One-l jar bioreactor, (2) 5-l jar bioreactor, (3) hollow-fiberr, (6) magnetic stirrer, (7–10) pump, (11) fresh medium reservoir, (12) permeateity controller.

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as cultured at 37 ◦C and pH 6.5. L. manihotivorans JCM 12514 was cultured at0 ◦C and pH 6.5. The culture pH was controlled by the addition of 5 M NaOH.o investigate optimum pH and temperature on direct l-lactic acid productionith soluble starch by the isolate No. 78, pHs were controlled between 5.5 and.0, and temperatures were between 30 and 45 ◦C. The batch fermentation set-p consisted of a simple pH-controlled system. In continuous fermentation, theystem was initially operated in batch mode and switched to continuous modefter the cultivation of 12 h.

.5. Cell-recycling and turbidostat

A hollow-fiber microfiltration module (MICROZA PSP 102, Asahi Kasei,okyo, Japan) was used to recycle cells back to the fermentor. The perfor-ance of this system was described previously [6]. The turbidity control

ystem consisted of a laser probe (LA-300LT, Automatic System Research,okyo, Japan) installed into the fermentor for online measurements of turbidity6].

.6. Continuous culture with cell-recycling and bleeding

The experimental set-up for the continuous culture with cell-recycling andleeding is shown in Fig. 1. In this study, Erlenmeyer flasks with a 400-ml totalorking volume and a 5-l bioreactor with a 4-l working volume were used for

he preculture and main culture, respectively. Initially, the batch culture wasonducted with MRS medium containing maltose (about 20 g l−1) statically forh until the cell growth of logarithmic growth middle phase. The culture was then

ransferred to the 1-l bioreactor by a pump. During the transfer, the broth in the-l bioreactor was recirculated through the microfiltration module by a pump, theermeate from the module was collected spontaneously, and a 4-l volume of broth

as finally concentrated to 0.4 l. After transfer and concentration, continuous

ulture was initiated by the feeding medium at an agitation rate of 150–200 rpmith cell-recycling. Further, for cell-recycling, the inflow of the feeding mediumas balanced by an outflow of the permeate from the module, while for cell-leeding the inflow of the feeding medium was balanced by an outflow of the

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able 1ugar fermentative test by API 50 CHL and data for E. faecium and E. durans

ugar No. 78 E. faecium E. durans

lycerol − d −rithritol − − −-Arabinose − ND ND-Arabinose + + −ibose + + −-Xylose − d −-Xylose − − −donitol − − −-Methyl-xyloside − − −alactose + + +-Glucose + + +-Fructose + + −-Mannose + + +-Sorbose − − −hamnose − − −ulcitol − − −

nositol − ND NDannitol + + −

orbitol − − −-Methyl-d-mannoside − ND ND-Methyl-d-glucoside − ND ND-Acetyl-glucosamine + + +mygdalin − + +rbutine + + +accharose + + drehalose + + +

+) Positive; (−) negative; ND: no data; (+) 75 to 89% are positive; d: discrepancies a

l Technology 41 (2007) 149–155 151

roth from the 1-l bioreactor. Therefore, the dilution rates (D) for this systemere calculated based on the following equations:

1 = F1

V, D2 = F2

V, D3 = F3

V, Dt = D1 + D2 + D3

here D1 is the dilution rate of the permeate (h−1), F1 the outflow rate (l h−1)f the permeate from the module, V the working volume (l) of the 1-l bioreactor,2 the dilution rate for addition of 5 M NaOH, F2 the outflow rate (1 h−1) of

he permeate, D3 the dilution rate for cell-bleeding (h−1), F3 the outflow ratel h−1) of the broth from the 1-l bioreactor and Dt is the total dilution rate (h−1)f this system. Unless otherwise stated, the cultivation conditions were the sames described above.

.7. Analysis

Cell density was measured in terms of optical density, at 562 nm, with a spec-rophotometer (UV–Visible Spectrophotometer BioSpec-1600, Shimadzu) andonverted to dry cell weight (DCW) using a calibration curve. The viable cellounts during fermentation were carried out on thioglycollate medium (Difco)ith 1.5% agar and indicated as a number of colony forming units per ml

CFU ml−1). Plates were incubated at 30 ◦C for 48 h. Glucose, maltose and lac-ic acid were determined with HPLC (US HPLC-1210, JASCO, Tokyo, Japan)quipped with a SUGAR SH 1011 column (Shodex, Tokyo, Japan) at 50 ◦Cith a 1.0 ml min−1 flow rate of 3 mM HClO4 as the mobile phase. Residual

tarch was determined by measuring the iodine-starch complex color [8]. Reduc-ng sugar was determined by Somogyi-Nelson method [13,14]. Total sugars inroth corresponding to starch and starch hydrolysis products including reduc-ng sugar were determined by enzymatic method [6]. Amylase activity wasssayed by determining degradation of soluble starch by iodine-starch complexolorimetric method. One enzyme unit (U) is defined as the amount of enzyme

hat permits the hydrolysis of 10 mg of starch for 30 min under the conditionsescribed by Nakamura [9]. Amylose content in starch was determined by amy-ose/amylopectin assay kit (Megazyme, Wicklow, Ireland) based on complexormation between the lectin concanavalin A and amylopectin. Optical purity ofactic acid produced was determined by the F-kit D/l-lactate assay (Roche Diag-

Sugar No. 78 E. faecium E. durans

Inulin − − −Melezitose − − −d-Raffinose + d −Amidon/starch + d dGlycogen + − −Xylitol − − −�-Gentibiose + ND NDd-Turanse − − −d-Lyxose − − −d-Tagatose − − −d-Fucose − − −l-Fucose − − −d-Arabitol − − −l-Arabitol − − −Esculine + ND NDSalicine + + +Cellobiose + + +Maltose + + +Lactose + + +Melobiose + (+) +5-Keto-gluconate − − −

mong reference studies.

152 K. Shibata et al. / Enzyme and Microbia

Table 2The effect of pH on direct l-lactic acid fermentation

pH Maximum lactic acid productionat the indicated time

YLA/TS (g g−1) PLA (g l−1 h−1)

g l−1 h

5.5 1.4 (±0.1) 76 0.54 (±0.07) 0.018 (±0.001)6.0 12.9 (±0.6) 40 0.70 (±0.04) 0.325 (±0.015)6.5 13.1 (±0.6) 33 0.76 (±0.05) 0.400 (±0.020)7.0 10.9 (±0.4) 26 0.54 (±0.05) 0.415 (±0.015)

YLA/TS: yield of lactic acid to consumed total sugar; PLA: lactic acid productivity(ac

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maximum lactic acid produced/ the indicated time). Cultivation was carried outt 30 ◦C with soluble starch (about 20 g l−1) as a substrate. The experiment wasarried out twice and the average data are shown with error.

ostic, Basel, Switzerland) with d-lactate dehydrogenase (LDH) and l-LDHctivity.

. Results and discussion

.1. Identification of the isolate No. 78

A partial 16S rDNA region of strain No. 78 was sequencednd analyzed. A sequence, corresponding to positions 8–519howed 99% identity to those of Enterococcus faecium and. durans. As shown in Table 1, sugar fermentation patternf strain No. 78 except utilization of several sugars includ-ng starch showed the highest similarity to that of E. faeciummong those of enterococcal species described by Manero andlanch [12]. Other characteristics such as chain cocci, catalase-

egative, growth at 10 ◦C, and homo l-lactic acid productionrom glucose, agreed with the criteria of Enterococcus [12].ccordingly, we concluded that the strain was identified as. faecium (BIOTECH 10375, Philippine National Collection

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able 3he effect of temperature on direct l-lactic acid fermentation

emperature (◦C) Maximum lactic acid production at the indicate

g l−1 h

0 9.4 (±0.9) 247 9.8 (±0.9) 273 1.5 (±0.2) 18

LA/TS: yield of lactic acid to consumed total sugar; PLA: lactic acid productivity (maH 6.5 with soluble starch (about 20 g l−1) as a substrate. The experiment was carried

able 4he effect of carbon source on lactic acid fermentation by E. faecium No. 78

arbon source Amylose (%) Maximum lactic acid production

g l−1 h

lucose – 18.6 (±0.5) 16oluble starch 17.6 15.4 (±0.9) 20ago starch 24.5 16.6 (±0.8) 15orn starch 53.3 13.2 (±1.2) 18heat starch 69.2 12.4 (±1.4) 21

otato starch 14.9 14.3 (±0.9) 24

LA/TS: yield of lactic acid to consumed total sugars, PLA: lactic acid productivity (mt pH 6.5 and 30 ◦C with the indicated carbon source (about 20 g l−1). The experimen

l Technology 41 (2007) 149–155

f Microorganisms), being a novel amylolytic lactic acid bac-erium.

.2. Optimal pH and temperature

Optimal pH was investigated by controlled between 5.5 and.0 at 30 ◦C. Little growth was observed at pH 5.5, leading toow lactic acid production. pH 6.5 provided better conditionhan the other pHs in terms of yield of lactic acid to consumedotal sugar (YLA/TS) and lactic acid productivity (PLA) (Table 2).ptimal temperature was investigated by controlled between 30

nd 45 ◦C at pH 6.5. Strain No. 78 did not show a good cellroliferation at 45 ◦C and produced the highest YLA/TS and PLAt 30 ◦C (Table 3). Therefore, optimal pH and temperature were.5 and 30 ◦C, respectively.

.3. Effect of different starches in direct l-lactic acidermentation

Glucose and several starches (e.g., soluble, sago, corn, wheatnd potato) were used as carbon sources for E. faecium No. 78 inRS medium. The stain No. 78 could produce l-lactic acid from

ll the starches tested, which resulted in different productionTable 4). The content of amylose of corn and wheat starches wasigher than those of the other starches. Starch containing highontent of amylose has a property of recrystallization of starchith decreasing temperature [15]. Therefore, it was considered

hat lactic acid production from corn and wheat starches wasower than that from sago starch, since amylase is difficult to

egrade the crystallized starch [16]. On the other hand, the chainength of amylopectin in sago starch was longer than that inotato starch [17]. Lactic acid fermentation may also depend onhe chain length of amylopectin in starch. As shown in Table 4,

d time YLA/TS (g g−1) PLA (g l−1 h−1)

0.78 (±0.04) 0.390 (±0.04)0.73 (±0.05) 0.365 (±0.04)0.74 (±0.05) 0.080 (±0.01)

ximum lactic acid produced/ the indicated time). Cultivation was carried out atout twice and the average data are shown with error.

at the indicated time YLA/TS (g g−1) PLA (g l−1 h−1)

0.83 (±0.03) 1.160 (±0.03)0.78 (±0.04) 0.770 (±0.05)0.93 (±0.02) 1.105 (±0.06)0.76 (±0.04) 0.735 (±0.07)0.68 (±0.05) 0.590 (±0.07)0.69 (±0.03) 0.595 (±0.04)

aximum lactic acid produced/ the indicated time). Cultivation was carried outt was carried out twice and the average data are shown with error.

K. Shibata et al. / Enzyme and Microbial Technology 41 (2007) 149–155 153

Fig. 2. Time course of direct lactic acid fermentation with sago starch. (A) E. faecium No. 78, (B) L. amylovorus JCM 1126, (C) L. manihotivorans JCM 12514,F withJ a arec

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ermentation was carried out at 200 rpm, pH 6.5 and 30 ◦C in a 1-l jar fermentorCM 1126 at 37 ◦C. This experiment was carried out twice and the average datell count, (�) reducing sugar and (�) amylase activity.

ago starch provided the highest PLA among all the starchesested. Moreover, PLA with sago starch was almost the same ashat with glucose as a conventional carbon source. Thus, sagotarch was superior to other starches as fermentation substrate forirect l-lactic acid production by employing E. faecium No.78.

.4. Comparison of direct lactic acid fermentation with E.aecium No. 78 and the other amylolytic lactic acid bacteria

Direct lactic acid fermentation with sago starch was carriedut using three amylolytic lactic acid bacteria, E. faecium No.8, L. amylovorus JCM 1126 and L. manihotivorans JCM 12514.s shown in Fig. 2, while starch concentration of L. amylovorus

CM 1126 and L. manihotivorans JCM 12514 decreased greatlyn early stages of cultivation, reducing sugar was produced at a

oderate concentration and decreased during production of lac-ic acid and acetic acid. In the case of E. faecium No. 78, starchoncentration was decreased gradually and reducing sugar andcetic acid were hardly detected (Fig. 2). The transient accu-

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rganism Starch Lactic acid (maximum, g l−1)

. faecium No. 78 Sago 16.6 (±0.5)

. amylovorus JCM 1126 Sago 14.3 (±0.9)

. mamhotivorans JCM 12514 Sago 11.0 (±0.8)

. amylovorus ATCC 33620 Cassava 4.8Potato 4.2

. plantarum Ogi El Soluble 8.0

. plantarum C5 Soluble 13.5

. plantarum OB8 Soluble 12.0

LA/TS: yield of lactic acid to consumed total sugars; PLA: lactic acid productivity (married out twice and the average data are shown with error.

400-ml medium (about 20 g l−1 of sago starch) except for that of L. amylovorusshown. (�) Starch, (©) total sugar, (�) lactic acid, (�) acetic acid, (�) viable

ulation of reducing sugar might partially result from differentctivities of amylases produced by the respective lactic acid bac-eria (Fig. 2). As shown in Table 5, E. faecium No. 78 providedighest values of maximum lactic acid production, YLA/TS andLA. Moreover, optical purity of l-lactic acid produced by E.

aecium No. 78 was 98.6% and higher than those by the othertrains. In addition, Table 5 indicates the results from this worknd from several literatures previously reported about final lacticcid concentration, YLA/TS and PLA in batch fermentation withtarches. E. faecium No. 78 is apparently an efficient lactic acidacterium for the direct fermentation.

.5. Continuous l-lactic acid fermentation withell-recycling and bleeding

There are few reports on continuous culture with amylolyticactic acid bacteria. In this study, the system was initiallyperated for 12 h in batch mode and then switched to con-inuous mode with different dilution rates. Table 6 shows the

bacteria

YLA/TS (g g−1) PLA (g l−1 h−1) Optical purity (%) Reference

0.93 (±0.02) 1.105 (±0.06) 98.6 This study0.81 (±0.05) 0.595 (±0.04) 85.4 This study0.56 (±0.08) 0.458 (±0.03) 83.7 This study0.48 0.69 – [18]0.42 0.14 – [18]0.31 0.48 – [19]0.71 0.56 – [20]0.63 0.50 – [20]

aximum lactic acid produced/ cultivation time). Experiments in this study were

154 K. Shibata et al. / Enzyme and Microbial Technology 41 (2007) 149–155

Table 6The parameter of continuous l-lactic acid fermentation with sago starch by E. faecium No. 78

Dilution rate VCC (Av, CFU/ml) Lactic acid (Av, g l−1) PLA (g l−1 h−1) Residual starch (Av, g l−1)

Continuous culture0.10 5.07 × l08 14.6 1.46 3.040.15 3.13 × l08 10.4 1.56 4.230.20 2.80 × l08 7.2 1.44 8.69

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inetic parameters of continuous cultures at several dilution rates0.10–0.20 h−1). PLA of continuous fermentation was higherhan that of batch culture (Tables 5 and 6). However, residualago starch concentration in the broth increased with increas-ng the dilution rates, which indicated that viable cell countecreased and starch degradation might be a rate-limiting stept higher dilution rate. These data indicated that the critical dilu-ion rate was probably 0.15 h−1 in the continuous system, athich volumetric lactic acid productivity was the maximum.56 g l−1 h−1 (Table 6).

To increase degradation rate of starch and to maintain aonstant cell concentration, continuous culture with high cellensity was carried out by cell-recycling and bleeding set-upepicted in Fig. 1. Cell-bleeding was conducted directly fromhe bioreactor after the total cell concentration reached approx-mately 45 g l−1, by balancing the inflow rate of the feeding

edium and the outflow rate of the broth. As described above,1, D2, D3 and Dt were applied to a continuous culture with cell-

ecycling and bleeding. To examine the effect of dilution rate inhis culture system, Dt was set at 0.16 and 0.26 h−1 (Fig. 3). Atoth the dilution rates, the starch concentration in the broth ofontinuous culture with high cell density was lower than thatf conventional continuous culture at Dt = 0.15 h−1 (Table 6).o glucose and maltose were detected in the reactor output.

s shown in Table 6, lactic acid produced and PLA at 0.16 h−1

f continuous fermentation with high cell density were higherhan those at 0.15 h−1 of conventional one, respectively. More-

ig. 3. Time course of continuous lactic acid fermentation with high cell densityt 30 ◦C and pH 6.5. (�) Starch, (©) total sugar, (�) lactic acid, (�) viable cellount and (�) dry cell weight.

[

2.86 1.913.04 3.24

ver, a maximum productivity of 3.04 g l−1 h−1 was achieved atdilution rate of 0.26 h−1 (average yield of lactic acid to con-

umed total sugar, 0.76 g g−1). This value was similar to thatf continuous lactic acid production from soluble starch usingmmobilized amylase and immobilized cells of Lactobacillusasei (Dt = 0.10 h−1) [21]. Thus, this study has shown an excel-ent potential for the production of lactic acid directly from sagotarch in a continuous fermentation with E. faecium No. 78.t should be especially emphasized that this system does notequire any kinds of pretreatment of starch for lactic acid fer-entation, such as enzymatic hydrolysis of starch by addition

f amylase.

cknowledgment

We thank Dr. Kopli B. Bujang of UNIMAS, Malaysia forindly providing sago starch.

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