Isolation and characterisation of lactic acid bacterium for effective ...

Appl Microbiol Biotechnol (2011) 89:1039–1049 DOI 10.1007/s00253-010-2986-4

BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING

Isolation and characterisation of lactic acid bacterium for effective fermentation of cellobiose into optically pure homo L-(+)-lactic acid Mohamed Ali Abdel-Rahman & Yukihiro Tashiro & Takeshi Zendo & Keisuke Shibata & Kenji Sonomoto

Received: 16 August 2010 / Revised: 25 October 2010 / Accepted: 25 October 2010 / Published online: 9 November 2010 # Springer-Verlag 2010

Abstract Effective utilisation of cellulosic biomasses for economical lactic acid production requires a microorganism with potential ability to utilise efficiently its major components, glucose and cellobiose. Amongst 631 strains isolated from different environmental samples, strain QU 25 produced high yields of L-(+)-lactic acid of high optical purity from cellobiose. The QU 25 strain was identified as Enterococcus mundtii based on its sugar fermentation pattern and 16S rDNA sequence. The production of lactate by fermentation was optimised for the E. mundtii QU25 strain. The optimal pH and temperature for batch culturing were found to be 7.0°C and 43°C, respectively. E. mundtii QU 25 was able to metabolise a mixture of glucose and M. A. Abdel-Rahman : T. Zendo : K. Shibata : K. Sonomoto (*) Laboratory of Microbial Technology, Division of Applied Molecular Microbiology and Biomass Chemistry, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan e-mail: [email protected] M. A. Abdel-Rahman Botany and Microbiology Department, Faculty of Science (Boys), Al-Azhar University, PN:11884 Naser City, Cairo, Egypt Y. Tashiro Department of Life Study, Seinan Jo Gakuin University Junior College, 1-3-5 Ibori, Kita-ku, Kokura, Kitakyushu City, Fukuoka 803-0835, Japan K. Sonomoto 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

cellobiose simultaneously without apparent carbon catabolite repression. Moreover, under the optimised culture conditions, production of optically pure L-lactic acid (99.9%) increased with increasing cellobiose concentrations. This indicates that E. mundtii QU 25 is a potential candidate for effective lactic acid production from cellulosic hydrolysate materials. Keywords L-Lactic acid production . Glucose . Cellobiose . Mixed sugars . Enterococcus mundtii

Introduction In recent decades, biotechnological production of optically pure lactic acid has become industrially paramount because of its cost-effectiveness and eco-friendliness. Specifically, highly optically pure lactic acid is necessary to produce highly crystalline poly-lactic acid, which endows polymers with high degrees of physical strength, and increased chemical and heat resistances (Lunt 1998). Optically pure lactic acid can be produced only through fermentation by microorganisms that can generate only one of its isomers, whereas synthetic production always results in a racemic mixture of lactic acid (Hofvendahl and Hahn-Hägerdal 2000). The demand for lactic acid has been increasing considerably owing to the promising applications of its polymers as an environmentally friendly alternative to petrochemicals plastics (Hujanen and Linko 1996). Cellulosic biomasses are inexpensive, abundant, renewable and readily available sources of sugar and they potentially meet the large lactic acid market demands (Wee et al. 2006). Biomass use provides less expensive raw materials, reduces the dependence on fossil fuels and helps in solving many environmental problems. However, direct lactic acid pro-

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duction from cellulose by lactic acid bacteria (LAB) has not been reported so far. The process of cellulose bioconversion to lactic acid is carried out in two main steps: first, hydrolysis of cellulosic material to glucose by chemical, physical or mostly enzymatic methods, and then lactic acid fermentation using the hydrolysate. Enzymatic methods depend on the synergistic actions of three cellulase components: endoglucanases (EGs), cellobiohydrolases (CBHs) and β-glucosidase (Nidetzky et al. 1994; Okano et al. 2010; Persson et al. 1991; Tolan and Foody 1999). Simultaneous saccharification and fermentation (SSF) of cellulose have been attempted by many authors (Abe and Takagi 1991; Bustos et al. 2005; John et al. 2006; Tanaka et al. 2006; Venkatesh 1997; Yáñez et al. 2003; Yoon 1997). In the SSF process, the commonly used cellulase from Trichoderma reesei has high EGs and CBHs activities, whereas the β-glucosidase activity is quite low (Stockton et al. 1991; Xia and Shen 2004). Thus, most of the intermediate products of cellobiose cannot be converted into glucose, and the accumulation of cellobiose will strongly inhibit EGs and CBHs activities (Fujii et al. 1995; Gusakov and Sinitsyn 1992; Holtzapple et al. 1990; Kruus et al. 1995; Medve et al. 1998). Therefore, effective hydrolysis of cellulose also requires highly active β-glucosidase, which breaks down cellobiose yielding two glucose molecules. Consequently, some researchers have suggested that improving β-glucosidase activity in the cellulase system is critical for raising hydrolysis yields (Shen and Xia 2004). In some studies, the cellulase complex was supplemented with βglucosidase to avoid cellobiose accumulation (Caminal et al. 1985; Moldes et al. 2001; Ramos and Saddler 1994). Glucose is also a strong inhibitor of cellulases (Adsul et al. 2007; Ghosh and Das 1971; Holtzapple et al. 1990). The potency of cellobiose inhibition is greater than that of glucose (Lee and Fan 1983). On the other hand, the presence of cellobiose in a fermentation medium could be interesting if this sugar is used as a carbon source in a subsequent fermentation stage. Because lactic acid fermentation of cellulose is challenging, rapid utilisation of cellobiose is of primary importance for efficient cellulose utilisation. However, to the best of our knowledge, there have been relatively few reports in the literature establishing lactic acid fermentation from cellobiose by using LAB strains, including Lactobacillus delberueki mutant Uc-3 (Adsul et al. 2007), Lactobacillus plantarum (Okano et al. 2010) and Lactobacillus lactis mutant RM2-24 (Joshi et al. 2010; Singhvi et al. 2010). Because all of these strains are mutant or recombinant strains, we attempted to isolate novel LAB strains that utilise major components of cellulosic hydrolysates efficiently. This would reduce the cost of lactic acid production from cellulosic materials by reducing the saccharifiying

Appl Microbiol Biotechnol (2011) 89:1039–1049

enzymes, specifically β-glucosidase. In the present study, we successfully isolated a novel LAB strain, Enterococcus mundtii QU 25 that could quickly ferment cellobiose to lactic acid at high concentration. Therefore, the cellulase reaction in the SSF process could be accelerated, and the bioconversion process from cellulosic materials to lactic acid could be improved.

Materials and methods Bacterial strains and media Modified Man, Rogosa and Sharpe (mMRS) medium was used for cell growth, inoculum preparation and fermentation as reported previously (Shibata et al. 2007), except that glucose (Nacalai Tesque, Kyoto, Japan) or cellobiose (Sigma, St. Louis, MO, USA) were used as carbon source for mMRS-glucose or mMRS-cellobiose media, respectively. Varying amounts of glucose and cellobiose were used as indicated in each experiment. The pH of the medium was adjusted to 7.0 using 1M NaOH or 1M HCl. CM-cellobiose agar medium contained the following (per litre): 5 g polypepton (Wako, Osaka, Japan), 5 g yeast extract (Nacalai Tesque), 5 g NaCl, 1 g cellobiose and 15 g agar supplemented with 0.5% CaCO3. The stock cultures of all bacterial strains were maintained at −80°C in 2 ml vials containing 30% glycerol until used. Isolation and screening for novel LAB Thirty environmental samples (soil, decayed wood, different pickles, plants and different animals’ fecal samples) were collected from various sources in Fukuoka City, Japan. Approximately 5 g of each was suspended in 100 ml of 0.85% NaCl, and 10 ml of this suspension was added to 100 ml mMRS broth in a 250 ml Erlenmeyer flask. The broths were enriched with 2% cellobiose. The flasks were incubated under anaerobic conditions at 30°C for 3–7 days, and then culture aliquots were spread evenly on CM cellobiose agar plates supplemented with 0.5% CaCO3 and incubated at 30°C. Bacterial colonies, which grew on the plates and formed a clear zone by acid formation, were individually picked and streaked on another plate. The isolates were maintained in mMRS cellobiose (1%) broth for immediate use or in 30% glycerol for storage at −80°C. Each of the isolates was first tested for catalase activity by placing a drop of 3% hydrogen-peroxide solution on the cells. Immediate formation of bubbles indicated the presence of catalase in the cells. Catalase-negative isolates were selected for further analysis. Cell morphology was observed by using phase-contrast microscopy at 1,000× magnification (Nikon Eclipse 80i, Tokyo, Japan).


These isolates were tested for several properties including fermentation profile, yield, optical purity and ability to grow and ferment cellobiose as a primary screening for lactic acid production. For this screening, fermentations were conducted in 15 ml screw-cap tubes filled with 9 ml mMRS medium enriched with 2% cellobiose. Inoculum (10%) was an overnight culture grown in mMRS cellobiose (1%). Fermentation profiles of the isolates were determined at 30°C after 24 h. Characterisation and identification of the QU 25 strain Physiological characteristics of the selected isolate was determined by using API 50 CHL test kit (bioMérieux, Marcy I’Etoile, France) as described by the manufacturer. The obtained pattern was compared with those of reference strains described by Manero and Blanch (1999). Partial 16S ribosomal deoxyribonucleic acid (16S rDNA) region of the isolate, corresponding to positions 8– 1510 of Escherichia coli 16S rDNA, was analysed using a set of universal primer 8UA and 1510r (Sawa et al. 2009). These sequences of the primers are as follows: 8UA, 5′AGAGTTTGATCCTGGCTCAG-3′; and 1510r, 5GGTTACCTTGTTACGACTT-3′. Total genomic DNA was extracted from cells treated with lysozyme (Seikagaku, Tokyo, Japan) using the Mag Extractor Kit (Toyobo, Osaka, Japan) according to the manufacturer’s protocol. Genomic DNA was used as a polymerase chain reaction (PCR) template. PCR was performed using Taq DNA polymerase (Promega, Madison, WI, USA) under the following conditions: denaturation at 94°C for 30 s, annealing at 50°C for 30 s and extension at 72°C for 90 s. The amplified products were purified using the QIAquick PCR purification kit (Qiagen, Hilden, Germany). DNA sequencing was carried out by FASMAC (Kanagawa, Japan). Similarity search was performed in the GenBank database using the BLAST algorithm. Analytical methods Cell growth was analysed by spectrophotometry (UV-1600 visible spectrophotometer, BioSpec, Shimadzu, Tokyo, Japan) at a wavelength of 562 nm. For the determination of dry cell weight (DCW), cells were centrifuged at 7,190×g for 20 min, washed twice with deionised water and dried at 105°C until a constant weight was achieved (24 h). The cell density was then converted to DCW (g/l) using an appropriate calibration curve. One unit of optical density at 562 nm corresponded to 0.218 g DCW/l. Cellobiose, glucose and fermentation products were determined using an high-performance liquid chromatography (HPLC) system (US HPLC-1210, Jasco, Tokyo, Japan) equipped with a SUGAR SH-1011 column (Shodex,

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Tokyo, Japan). Aliquots of the sample cultures (1 ml) were centrifuged at 2,000×g for 10 min at 4°C. Thereafter, the supernatant was diluted in ultrapure water, filtered using a membrane filter (Dismic-13HP, 0.45 μm, Advantec, Tokyo, Japan) and finally injected in the HPLC system under the following conditions: column temperature of 50°C, 3 mM HClO4 as the mobile phase at a flow rate of 1.0 ml/min and an injection volume of 20 μl. The concentrations of residual sugars and fermentation products were calculated using calibration curves obtained using standard solutions. The optical purity of lactic acid was measured using a BF-5 biosensor (Oji Scientific Instruments, Hyogo, Japan) according to the manufacturer’s protocol. Enzyme assay Aryl-β-glucosidase and aryl-β-cellobiosidase activities were measured as described previously (Eberhart 1961; Adsul et al. 2007) with 5 mM p-nitrophenyl-β-Dglucopyranoside (Sigma) and p-nitrophenyl-β-D-cellobioside (Sigma) as substrates, respectively. The hydrolysis of substrates was carried out in 50 mM citrate–phosphate buffer (pH 7.0) at 43°C for 30 min. Assay mixture (1 ml) consists of 0.9 ml of substrate and 0.1 ml of cell suspension or culture supernatant shown in result section. Absorbance was measured spectrophotometrically at 410 nm, and the liberated p-nitrophenol concentration was inferred from p-nitrophenol standard. One unit of enzyme activity is equivalent to 1 μmol of p-nitrophenol generated per minute. Fermentation parameters The fermentation parameters evaluated in this study were the specific growth rate (μ), calculated as follows: μ (h−1)= ln (x2÷x1)÷(t2−t1), where x is OD562 and t is the sampling time (h). The yield of lactic acid based on substrate consumed (Y, g/g) was defined as the ratio of lactic acid produced (g/l) to amount of sugar consumed (g/l). Lactic acid productivity (g/l/h) was calculated as the ratio of lactic acid concentration (g/l) to the fermentation time (h). Maximum lactic acid productivity was calculated between each sampling periods within exponential growth phase. Purity of L-lactic acid was measured as follows: optical purity (%) = (L-lactic acid concentration–D-lactic acid concentration) ÷ (L-lactic acid concentration + D-lactic acid concentration) × 100. Flask fermentation Time-course fermentation by QU 25 strain was carried out in 200 ml Erlenmeyer flasks in a working volume of 100 ml mMRS cellobiose (2%). Inoculum (10%) was an overnight

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culture grown in the same medium. Fermentation profiles of the QU 25 strain were determined at 30°C and initial pH 6.5. Samples were removed at desired intervals for determination of pH, biomass (DCW), residual sugars and fermentation products. Fermentor-type fermentations Inocula preparations were as follows: 1 ml of glycerol stock was used to provide inocula for tubes containing 9 ml mMRS sugar (1%) in 15 ml screw-cap tubes and incubated for 24 h. Of this culture, 4 ml was then transferred to a new growth medium in a 50 ml screw-cap tube that contained 36 ml of the same medium. The inoculum was incubated at the desired temperature for 8 h before inoculation at 10% in the jar fermentor. Fermentations were carried under agitation at 200 rpm in a 1 l jar fermentor (Biott, Tokyo, Japan) containing a working volume of 400 ml mMRS supplemented with desired sugar concentrations. Sugar solution was autoclaved separately and mixed with the rest of mMRS media. The inoculum (10%) was derived from a culture grown on the same medium supplemented with 1% sugar. The samples were withdrawn at desired intervals and frozen at −20°C for further analysis. At each sampling time, 5 ml of the cultures were removed aseptically from each jar and assayed for biomass (DCW), residual sugars and fermentation products. To investigate the optimal pH, fermentations were performed without or with controlling pH by addition of 5 M NaOH using a peristaltic pump connected to an automatic pH controller (PHC-2201, Biott, Tokyo). pH was controlled at 5.5, 6.0, 6.5, 7.0 or 7.5, respectively. Fermentations were carried out in mMRS cellobiose (2%). Fermentation profile of the strain was determined at 30°C. To investigate the effect of temperature on lactic acid production, fermentations using mMRS cellobiose (2%) were performed at different temperatures (30°C, 37°C, 43°C, 45°C, 47°C or 50°C) under controlled pH at 7.0 as described above. The effect of sugar mixture was studied using glucose/ cellobiose at a ratio 1:1 (w/w) at 10, 15 or 20 g/l each. Inoculum was 10% of mMRS cellobiose (1%) as described above. Fermentations were carried out at 43°C under controlled pH at 7.0. The effect of substrate concentration was studied by varying the cellobiose concentration in mMRS cellobiose broth at 50.8, 101 or 151 g/l. Fermentations were carried out at 43°C. pH was controlled at 7.0 by automatic addition of 10 M NaOH for initial cellobiose concentration of 50.8 and 101 g/l and 15 M NaOH for 151 g/l. At initial cellobiose concentration of 151 g/l, the fermentors were supplemented with 1%, 0.25% and


0.25% of yeast extract after 8, 60 and 96 h incubation, respectively. Nucleotide sequence accession number 16S rDNA sequence determined for this study has been deposited in the DNA Data Base of Japan (DDBJ) under accession no. AB576587.

Results LAB isolation and screening In the first stage of this work, 631 bacterial isolates were obtained from 30 environmental samples according to the procedures described in ‘Materials and methods’. A total of 253 amongst these isolates were catalase-negative and showed clear zone by acid formation on CM cellobiose agar plates supplemented with CaCO3. These isolates were tested for several properties such as fermentation profile, lactic acid yield, type of lactic acid isomer produced and ability to grow and ferment cellobiose. Based on the results of these experiments, strain QU 25 performed better than the other isolates in yield (approx. 1 g/g consumed sugar) and optical purity (≥99.9%) of lactic acid. This isolate was then selected for detailed study. Strain QU 25 was isolated at 30°C from ovine fecal samples collected from Fukuoka-Zoo, Japan, and exhibited cocci-shaped morphology. Characterisation and identification of strain QU 25 The selected strain was characterised by bacterial identification kit of API-CHL, which tests the acid formation from various sugars and sugar alcohol. As shown in Table 1, the sugar fermentation pattern of strain QU 25, excluding utilisation of D-tagatose, showed the highest similarity to those of E. mundtii and E. casseliflavus amongst those of enterococcal species described by Manero and Blanch (1999). Other characteristics, such as coccal morphology, catalase negativity, growth at 45°C and homo L-lactic acid production from glucose, agreed with characteristics of Enterococcus (Manero and Blanch 1999). A partial 16S rDNA region of strain QU 25 was sequenced and analysed. A sequence, corresponding to positions 8–1,510 bp, showed 99% identity to that of reference strain E. mundtii SS1232 (accession number GQ337033.1) available in the NCBI (http://www.ncbi.nlm.nih.gov). Accordingly, we concluded that the strain QU 25 was identified as E. mundtii [deposited at NITE Biological Resource Center (NBCR), National Institute of Technology and Evaluation, Tokyo, Japan, as NITE BP-965].


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Table 1 Sugar fermentation pattern of isolate QU 25 by API 50 CHL and data for Enterococcus mundtii and Enterococcus casseliflavus Sugar

QU 25

E. mundtii

E. casseliflavus

Sugar

QU 25

E. mundtii

E. casseliflavus

Glycerol Erythriol D-Arabinose L-Arabinose D-Ribose D-Xylose L-Xylose Adonitol

± – – + + + – –

d – ND + + + – –

V – ND + + + – –

Salicin Cellobiose Maltose Lactose Melibiose Sucrose Trehalose Inulin

+ + + + + + + –

+ + + + + + + d

+ + + + + + + (+)

β-Methyl-D-xyloside Galactose D-Glucose D-Fructose Mannose L-Sorbose Rhamnose Dulcitol Inositol Mannitol Sorbitol α-Methyl-D-mannoside α-Methyl-D-glucoside N-Acetyl-glucosamine Amygdalin Arbutin Esculin

– + + + + – + – – + + – – + + + +

– + + + + – + – ND + d (+) – + + + +

– + + + + – (+) – ND + d V + + + + +

Melezitose D-Raffinose Starch Glycogen Xylitol α-Gentibiose D-Turanose D-Lyxose D-Tagatose D-Fucose L-Fucose D-Arabitol L-Arabitol Gluconate 2-Keto-gluconate 5-Keto-gluconate

– – ± – – + – – + – – – – – – –

d (+) d (−) – ND – – – – – – – – – –

D D V – – ND V – – – – – – + – –

+ Positive, (+) 75–89% are positive, V 26–74% are positive, (−) 11–25% are positive,–negative, d discrepancies among reference studies, ND no data, ± weak fermentation

Optimal pH and temperature for lactic acid fermentation from cellobiose Subsequently, the time-course study was conducted to examine the growth and lactic acid producing capacity of E. mundtii QU 25. From fermentations conducted in Erlenmeyer flasks, we found that growth of strain QU 25 levelled off at about 0.91 g/l DCW after 21 h, pH dropped to 4.85, and maximum L-(+)-lactic acid production was 7.04 g/l after 60 h of incubation with very low amount of by-products. In order to study the effect of pH on lactic acid production, fermentations were carried out under controlled pH at 5.5, 6.0, 6.5, 7.0 or 7.5 to verify the possibility of increasing lactic acid production. Table 2 shows kinetic parameters of batch fermentations at different pH values. In these fermentations, HPLC analysis showed that fermentation was a homolactic process because no other peaks related to other components appeared except for a very small amount of acetate as a by-product. The low pH of 5.5–6.0 greatly inhibited cell growth, whereas the higher pH values from 6.5 to 7.5

were conducive to fast bacterial growth. At pH values from 5.5 to 6.0, cellobiose consumption was very slow and it did not deplete until the end of fermentation. At pH values of 6.5–7.5, the growth curve passed a quick growth phase followed by a relatively long stationary phase (about 12–16 h). The maximal lactic acid productivity increased with pH, with the highest value of 2.2 g/l/h at pH 7.5, the biomass was approximately 4.5-fold higher at pH 7.5 than non-pH-controlled fermentation. Cellobiose consumption rates were higher at pH values between 7.0 and 7.5 in comparison to that at lower pH values (data not shown). Formation of lactic acid continued until cellobiose was depleted. Although the maximum lactic acid productivity and biomass were the highest at pH 7.5, the concentration and yield of lactic acid did not exhibit maximum values. After 20-h cultivation duration, the concentration, yield and optical purity of L-lactic acid at pH 7.0 were 18.7 g/l, 0.94 g/g and 100%, respectively. At pH 7.0, the produced lactic acid and the yield increased by 376% and 43%, respectively, in comparison to non-pHcontrolled batches (Table 2).

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Table 2 Effects of pH of culture medium on lactic acid fermentation from cellobiose by 1 Enterococcus mundtii QU 25 pH

μmaxa (h−1)

Maximum cell mass (g/l)

Maximum L-lactic acid (g/l) produced at indicated time

L-lactic

No control 5.5 6.0 6.5 7.0 7.5

0.415 0.368 0.610 0.672 0.635 0.575

0.71 1.38 1.89 2.37 3.04 3.17

3.90 13.9 14.8 14.7 18.7 16.4

0.657 0.875 0.840 0.866 0.945 0.831

±0.09 ±0.76 ±0.89 ±0.00 ±2.04 ±0.25

(24 (24 (24 (20 (20 (20

h) h) h) h) h) h)

acid yieldb (g/g)

Maximum L-lactic acid productivity (g/l/h) 0.47 0.82 1.48 1.81 2.10 2.20

Fermentations were carried out at 30°C and the indicated pH with initial cellobiose concentration 20 g/l. Averages with standard deviations are based on three independent fermentations a

Maximum specific growth rate

b

Produced lactic acid (g/l) per consumed sugar (g/l)

We carried out further assays at different fermentation temperatures ranging from 30°C to 50°C (Table 3). QU 25 showed good biological activity producing high concentrations and yields of lactic acid over a wide range of temperature from 30°C to 45°C. At 43°C, all cellobiose was utilised after 20 h and ca. 20 g/l lactic acid was produced with a yield of ~1.0 g/g consumed cellobiose. Glucose was not observed in the medium during fermentation of cellobiose. Maximal lactic acid productivity and maximum growth rate increased with increasing temperature with the respective highest values of 3.44 g/l/h and 0.675 h−1 at 43°C (Table 3). At temperatures higher than 45°C, strain QU 25 did not show either good cell proliferation or high lactic acid production. Thus, the optimum temperature was 43°C. Measurement of aryl-β-glucosidase and aryl-β-cellobiosidase activities E. mundtii QU 25 cells were grown with glucose and cellobiose and harvested at late exponential phase by centrifugation. The cells were washed three times with citrate

phosphate buffer (50 mM, pH 7.0) and suspended in the same buffer. This suspension was used for analysing the intracellular activities of aryl-β-glucosidase and aryl-β-cellobiosidase. Higher activities were detected with cellobiose-grown cell suspension one (aryl-β-glucosidase, 25.7 U/mg of DCW; aryl-β-cellobiosidase, 8.95 U/mg of DCW) rather than glucose-grown cells (aryl-β-glucosidase, 0.864 U/mg of DCW; aryl-β-cellobiosidase, 0.813 U/mg of DCW). However, no enzyme activities were detected in the culture supernatant obtained by centrifugation of the cultured broth. Lactic acid fermentation with sugar mixture by E. mundtii QU 25 In order to study the effect of sugar mixtures on lactic acid fermentation, QU 25 was cultivated in fermentation media with different glucose/cellobiose concentrations. Figure 1 shows the sugar consumption and lactic acid production at final sugar concentration of 20 g/l. Interestingly, E. mundtii QU 25 consumed both glucose and cellobiose simultaneously within 6 h of fermentation. The sugar consumption

Table 3 Effects of temperature on lactic acid fermentation from cellobiose by Enterococcus mundtii QU 25 Temperature (°C)

μmaxa (h−1)


Maximum L-lactic acid produced (g/l) at indicated time

L-Lactic

acid yieldb (g/g)

Maximum L-lactic acid productivity (g/l/h)

30 37 43 45 47 50

0.640 0.662 0.675 0.400 0.200 0.136

3.17 2.88 2.64 1.72 0.52 0.36

20.3 20.2 20.4 17.9 5.80 3.05

1.03 1.03 1.04 0.93 1.03 0.60

2.54 3.07 3.44 1.77 0.46 0.30

±0.57 ±0.76 ±0.63 ±0.17 ±0.02 ±0.09

(20 (20 (20 (16 (24 (24

h) h) h) h) h) h)

Fermentations were carried out with controlled pH at 7.0 and the indicated temperature with initial cellobiose concentration 20 g/l. Averages with standard deviations are based on three independent fermentations a


b


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Dry cell weight (g/l)

L-Lactic acid and sugars (g/l)


cellobiose concentrations, there was a very short lag phase. After the lag phase, for all cases, the cell growth rates were more or less the same (0.740–0.785 h−1) in the exponential phases (Table 5). Maximum concentration of L-lactic acid increased with increasing cellobiose concentration up to 119 from 151 g/l cellobiose, the optical purity of which was 99.9% (Table 5). Figure 2 depicts lactic acid fermentation from 151 g/l cellobiose, showing no glucose formation and very little formation of by-products (acetic acid or ethanol) during the fermentation.

Discussion

Fig. 1 Lactic acid fermentation by Enterococcus mundtii QU 25 using a glucose/cellobiose mixture at 10 g/l each. Fermentation was carried out at 200 rpm, pH 7.0 and 43°C in a 1 l jar fermentor containing 400 ml mMRS. Symbols: ○ dry cell weight, ● L-lactic acid, Δ glucose and ▲ cellobiose. Data points represent the means and standard deviations of results from three independent experiments. The standard deviation is less than the size of symbols if no error bars are seen

rates were almost similar for glucose and cellobiose with 1.69 and 1.53 g/l/h, respectively. The final L-lactic acid concentration increased with increasing sugar concentration up to 40 g/l with almost similar lactic acid yields (Table 4). No by-products, such as acetic acid or ethanol, were observed in all fermentations. High lactic acid production from cellobiose by E. mundtii QU 25 In order to demonstrate the effects of cellobiose concentration on growth of QU 25 strain and to obtain high lactic acid concentrations, we conducted fermentation assays using three levels of initial cellobiose concentrations (ca. 50.8–151 g/l) at 43°C and pH 7.0. Interestingly, at all the

Cellulosic biomass is a potential feedstock for lactic acid production technology receiving much attention due to its feasibility and valuable products. Because LAB lack a cellulase system (Tokuhiro et al. 2008), many technologies have been developed for pretreatment and fermentation of cellulose including the SSF process. However, much expensive cellulases are used due to enzyme inhibition with products such as cellobiose. Therefore, effective lactic acid production from cellulosic materials requires microorganisms that potentially utilise cellulose hydrolysates, such as cellobiose, efficiently affording an economically feasible process. The main objective of the present study was to isolate a potential bacterial strain for efficient utilisation and fermentation of cellulosic components in order to increase the cellulose-saccharifiying rate during the SSF process and consequently to increase lactic acid production and to reduce capital production costs (Anuradha et al. 1999). Since the isolation and screening of microorganisms from natural sources has always been the most powerful means of obtaining useful and genetically stable strains for industrially important products (Adnan and Tan 2007), we obtained 253 bacterial isolates from 30 different environmental samples collected from Fukuoka City, Japan. These isolates were determined preliminarily as LAB, which

Table 4 Lactic acid fermentation with sugar mixture of glucose and cellobiose by Enterococcus mundtii QU 25 Carbon source (g/l) Glucose

Cellobiose

10 15 20

10 15 20

μmaxa (h−1)

0.668 0.850 0.620


Maximum L-lactic acid (g/l) produced at indicated time

L-Lactic

3.04 3.40 3.62

18.9±0.26 (6 h) 26.9±0.02 (10 h) 35.1±0.10 (15 h)

0.970±0.036 0.960±0.001 0.913±0.004

yield

b

acid (g/g)

Average L-lactic acid productivityc (g/l/h)

3.15 2.58 2.99

Fermentations were carried out at 43°C and controlled pH at 7.0. Averages with standard deviations are based on three independent fermentations a


b


c

During the exponential growth

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Table 5 Effect of cellobiose concentrations on lactic acid fermentation by Enterococcus mundtii QU 25 Cellobiose concentration (g/l)

μmaxa (h−1)


Maximum L-lactic acid produced (g/l) at indicated time

L-lactic acid yieldb (g/g)

Maximum L-lactic acid productivity (g/l/h)

Optical purity of L-lactic acid (%)

50.8 101 151

0.785 0.777 0.740

2.89 2.88 5.47

50.1±0.82 (20 h) 89.8±1.00 (72 h) 119±3.70 (106 h)

1.02 0.913 0.830

3.53 3.69 4.50

99.9 99.9 99.9

Fermentations were carried out at 43°C and controlled pH at 7.0. Averages with standard deviations are based on three independent fermentations a


b


Dry cell weight (g/l)

Cellobiose and fermentation products (g/l)

produced lactic acid from cellobiose. Amongst the cellobiose-utilising LAB, isolate QU 25 performed better than the others in lactic acid yield from cellobiose, produced L-(+)-lactic acid with an optical purity of ≥99.9% and therefore was selected for further study. The usual ecological niche for Enterococcus species is the intestine of humans and other animals (Manero and Blanch 1999). E. mundtii was discovered in 1986 as a nonmotile, yellow pigmented enterococcus isolated from soil, milkers’ hands, cow teats and plants (Collins et al. 1986). This species has been rarely isolated from environmental sources (Junco et al. 2001), but we have previously isolated bacteriocin-producing E. mundtii QU 2 from soybean (Zendo et al. 2005).

Time (h)

Fig. 2 Lactic acid fermentation from 151 g/l cellobiose by Enterococcus mundtii QU 25. Fermentations were carried out at 200 rpm, pH 7.0 and 43°C in a 1 l jar fermentor containing 200 ml mMRS. The fermentors were supplemented with 1%, 0.25% and 0.25% of yeast extract after 8, 60 and 96 h incubation, respectively. Symbols: ○ dry cell weight, ● L-lactic acid, Δ acetic acid, ▲ ethanol and □ cellobiose. Data points represent the means and standard deviations of results from three independent experiments. The standard deviation is less than the size of symbols if no error bars are seen

Controlling the pH allowed the final biomass and lactic acid concentration to increase markedly (Table 2). Interestingly, QU 25 showed lactate fermentation efficiency at a wide range of temperatures (30–45°C) and produced Llactic acid with high production rate at higher temperatures, specifically at 43°C (Table 3). Increasing growth rate at early fermentation stages at 43°C resulted in higher lactic acid production and higher volumetric productivity of 20.4 g/l and 3.44 g/l/h, respectively. Romero-Garcia et al. (2009) reported 13.0 g/l of L-lactic acid from cellobiose utilisation with relatively low productivity of 0.36 g/l/h and an optical purity of 99.5% by using Bacillus subtilis CH1, a genetically modified strain of B. subtilis, at 37°C and pH 7.0. Our strain, QU 25, produced only lactic acid as a major fermentation product. Recently, Okano et al. (2010) constructed two genetically modified strains of L. plantarum NCIMB 8826, pCU/ΔldhL1 and pCU-CelA/ΔldhL1, which produced 2.13 and 1.99 g/l D-lactic acid, and 0.33 and 0.30 g/l acetic acid from 2.69 and 2.41 g/l cellobiose, respectively, at 37°C. Moreover, lactic acid produced by these strains is temporarily accumulated in the culture and then converted to acetic acid with formation of 2.02 and 2.14 g/l acetic acid by pCU/ΔldhL1 and pCU-CelA/ΔldhL1, respectively. Furthermore, the ability of QU 25 to grow at high temperatures is a desirable trait since a high fermentation temperature reduces contamination by other microorganisms (Tsai et al. 1993). The cellulose saccharification process yields carbohydrate feedstocks containing a variety of sugars. In order to maximise lactic acid yield and production, complete utilisation of mixed sugars is essential. Carbon catabolite repression (CCR) is a common phenomenon in LAB accomplished by a complex regulatory mechanism (Kim et al. 2009). Very few LAB have been reported which consume different sugars simultaneously. L. brevis cometabolises glucose/xylose, glucose/arabinose and glucose/xylose/arabinose simultaneously (Kim et al. 2010). When a CCR-positive strain is used for lactic acid production from mixed sugar substrates, the overall process design is restricted and productivity is reduced because of


sequential utilisation of the sugars. Therefore, for industrialisation of lactic acid production from cellulosic materials, we studied the efficiency of E. mundtii QU 25 for utilisation of glucose/cellobiose mixture as the main sugars present in cellulose hydrolysates. Simultaneous fermentation of mixed carbohydrates by E. mundtii QU 25 exhibited utilisation rates and fermentation behaviour similar to those seen when using as a single carbon source. At all initial sugar concentrations, our strain showed simultaneous utilisation patterns that is strongly indicative of no CCR by glucose (Fig. 1; Table 4). To the best of our knowledge, no LAB strain demonstrating efficient simultaneous utilisation of cellobiose and glucose has been reported previously. L-Lactic acid fermentation by E. mundtii QU 25 with cellobiose as substrate has been shown to be a highly growth-associated process. High substrate concentrations may generally inhibit cell growth. In order to explore the effects of initial substrate concentration on cell growth, three experiments using mMRS broths with initial cellobiose concentrations ranging from ca. 50.8 to 151 g/l were carried out at 43°C and pH 7.0 (Table 5; Fig. 2). Maximal growth rate was almost similar at any cellobiose concentration, which indicates no substrate inhibition up to 151 g/l. Maximal DCW was similar at cellobiose concentration of 50.8–101 g/l, whereas the higher growth at 151 g/l cellobiose could probably be due to supplementation of yeast extract during fermentation. It can be concluded that substrate inhibitory effects in lactic acid fermentation may be negligible. It was observed that, as the cellobiose concentration is higher, there is an increase in maximum lactic acid productivity (Table 5) as well as a remarkable increase in cellobiose consumption rate (data not shown). Conversely, the yield of lactic acid was decreased with increasing cellobiose concentration (Table 5), which may be due to product inhibition or exhaustion of one restricting nutrient or the combined effect of both. These positive and negative correlations in terms of maximum productivity with sugar consumption rate and yield of lactic acid, respectively, also have been previously reported by Tokuhiro et al. (2008). The optical purity of L-lactic acid was 99.9% at all cellobiose concentrations. Our results indicate that capabilities of QU 25 to utilise cellobiose and highly resist osmotic stress caused by high substrate concentration are highly advantageous. Many process engineering techniques, such as substrate fed-batch systems, have been developed to decrease osmotic stress inhibition in lactic acid fermentation (Ding and Tan 2006). In a previous report, Adsul et al. (2007) achieved a production level of 90 g/l L-lactic acid from cellobiose using Lactobacillus delbrueckii mutant Uc-3 in a batch process, but did not determine the optical purity of the product. The central problem with lactic acid production in

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Lactobacillus spp. has been the optical purity of the product being less than 95%, because Lactobacilli have both L- and D-lactate dehydrogenases (Hofvendahl and Hahn-Hägerdal 2000; Romero-Garcia et al. 2009; Saitoh et al. 2005). Therefore, 5% content of isomeric impurities of L-lactic acid may increase the costs of purification processes, which are already complex (Romero-Garcia et al. 2009). Severson and Barrett (1995) reported that the parent strain Lactobacillus bulgaricus, strain R-160 Biolac™, typically produces 70% L -(+)-lactic acid and 30% D-(-)-lactic acid. L. bulgaricus has been classified as L. delbrueckii ssp. bulgaricus (Severson and Barrett 1995). It was also reported that Lactobacillus sp. RKY2 is a homofermentative LAB and produces DL-lactic acid from glucose with an optical purity of 70% L-(+)-lactic acid (Wee et al. 2005; Yun et al. 2004). Recently, the mutant RM 2-24 of L. lactis was shown to produce a maximum concentration of 80 g/l D-lactic acid from 100 g/l cellobiose with an optical purity of 98% (Joshi et al. 2010; Singhvi et al. 2010). Our strain, QU 25, produced higher concentration of L-lactic acid with higher yield and an extremely high optical purity (≥99.9%). However, it was reported that few LAB could produce optically pure lactic acid (Benthin and Villadsen 1995) and isomeric purity of lactic acid changes with pH, temperature and substrate concentration using microbial fermentation by a single strain of LAB (Hofvendahl and Hahn-Hägerdal 1997; Zhang et al. 2008). Interestingly we observed no significant change in optical purity with our strain during fermentation experiments. In all fermentation experiments with cellobiose as the sole carbon source, glucose was not detected in fermentation broth at any time, indicating that hydrolytic enzymes are not extracellular. We measured these enzymes in glucose- and cellobiose-grown cells. We could not detect any enzyme activity in the culture supernatant obtained by centrifugation of the cultured broth, suggesting that these enzymes are intracellular. Higher levels of enzyme activities were observed in cellobiose-grown cells than glucosegrown cells, indicating that these enzymes are inducible. Aryl-β-glucosidase was reported as a cell wall/cell membrane-bound enzyme in L. delberueki mutant Uc-3 (Adsul et al. 2007) and L. lactis mutant RM2-24 (Singhvi et al. 2010). In conclusion, E. mundtii QU 25 proved to be a promising strain enabling production of L-lactic acid from cellobiose. Using this strain, bottlenecks and feedback inhibition by cellobiose will be removed, leading to nearly complete conversion of cellulosic substrates to L-lactic acid. From an economical point of view, using QU 25, the cost of lactic acid production from cellulosic materials will be reduced due to an expected decrease in enzyme requirements, a significant cost component in the use of biomasses for production of value-added products. This study shows

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the potential of such a strain in reaching the demand for lactic acid, meeting its widest applications nowadays. It should be emphasised that this is the first report on simultaneous utilisation of glucose and cellobiose by a LAB strain. Acknowledgment M.A. Abdel-Rahman is supported by an Egyptian government scholarship offered by the Ministry of Higher Education and Scientific Research, Egypt.

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