FEMS Microbiology Letters, 362, 2015, 1–7 doi: 10.1093/femsle/fnu030 Advance Access Publication Date: 4 December 2014 Research Letter
R E S E A R C H L E T T E R – Biotechnology & Synthetic Biology
Enterococcus faecium QU 50: a novel thermophilic lactic acid bacterium for high-yield l-lactic acid production from xylose Mohamed Ali Abdel-Rahman1,2 , Yukihiro Tashiro3,4 , Takeshi Zendo1 , Kenji Sakai3 and Kenji Sonomoto1,5,∗ 1
Laboratory of Microbial Technology, Division of Systems Bioengineering, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan, 2 Botany and Microbiology Department, Faculty of Science (Boys branch), Al-Azhar University, PN:11884, Nasr City, Cairo, Egypt, 3 Laboratory of Soil Microbiology, Division of Systems Bioengineering, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, Japan, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan, 4 Institute of Advanced Study, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan and 5 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 ∗ Corresponding author: Laboratory of Microbial Technology, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan. Tel: +81-(0)92-642-3019; Fax: +81-(0)92-642-3019; E-mail: [email protected] One-sentence summary: A novel thermophilic l-lactic acid bacterium, Enterococcus faecium QU 50, was isolated. Strain QU 50 showed high lactic acid yield (∼1.0 g g−1 ) from several lignocellulose-derived sugars. QU 50 produced l-lactic acid homofermentatively (∼1.0 g g−1 yield) from low-concentration xylose. QU 50 showed efficient fermentation at 50◦ C. QU 50 is the only thermophilic LAB strain that utilizes hexose and pentose sugars homofermentatively. Editor: Michael Sauer
ABSTRACT Production of optically pure lactic acid from lignocellulosic material for commercial purposes is hampered by several difficulties, including heterofermentation of pentose sugars and high energy consumption by mesophilic lactic acid bacteria. Here, we report a novel lactic acid bacterium, strain QU 50, that has the potential to produce optically pure l-lactic acid (≥99.2%) in a homofermentative manner from xylose under thermophilic conditions. Strain QU 50 was isolated from Egyptian fertile soil and identified as Enterococcus faecium QU 50 by analyzing its sugar fermentation pattern and 16S rRNA gene sequence. Enterococcus faecium QU 50 fermented xylose efficiently to produce lactic acid over wide pH (6.0–10.0) and temperature ranges (30–52◦ C), with a pH of 6.5 and temperature of 50◦ C being optimal. To our knowledge, this is the first report of homofermentative lactic acid production from xylose by a thermophilic lactic acid bacterium. Key words: Lactate fermentation; biomass utilization; biorefinery; pentose; homofermenters; high temperature
Received: 7 November 2014; Accepted: 11 November 2014 C FEMS 2014. All rights reserved. For permissions, please e-mail: [email protected]
1
2
FEMS Microbiology Letters, 2015, Vol. 362, No. 1
INTRODUCTION Optically pure lactic acid (LA) has garnered increasing attention due to its high potential for a wide range of applications, mainly in food, chemical, cosmetic and pharmaceutical industries. Optically pure LA can only be produced by microbial fermentation because chemical synthesis produces racemic dl-LA (Tashiro et al., 2011). The main microbial LA producers are bacteria and fungi, although the latter exhibits relatively lower productivity and needs complicated requirements for LA production (Abdel-Rahman, Tashiro and Sonomoto 2013a). Bacterial producers are classified into four main groups: lactic acid bacteria (LAB), Bacillus strains, Escherichia coli and Corynebacterium glutamicum. Unlike the other bacterial producers, LAB produce LA as an anaerobic product of glycolysis with high yield and productivity. LAB are characterized as Gram-positive, non-sporing, catalase-negative, mesophilic (10–45◦ C) but aerotolerant cocci or rods that cannot use oxygen as an electron acceptor during sugar fermentation. Therefore, LAB have not included other LA producers such as Bacillus strains and some filamentous fungi (Carr, Chill and Maida 2002). LAB are comprised of about 20 genera among which the most important are Lactococcus, Enterococcus, Lactobacillus, Streptococcus, Leuconostoc, Pediococcus, Carnobacterium, Oenococcus, Aerococcus, Tetragenococcus, Weissella and Vagococcus (Reddy et al., 2008; Abdel-Rahman, Tashiro and Sonomoto 2013a). Currently, lignocellulosic biomasses are considered as an economically attractive feedstock for LA production, which does not significantly affect food supply and price. Lignocellulose is composed of cellulose (35–50%), hemicellulose (20– 40%) and lignin (10–30%); thus, it contains large amounts of fermentable sugars in the form of glucose and xylose (AbdelRahman, Tashiro and Sonomoto 2011a, 2013a). Xylose is the second most abundant sugar in nature, next to glucose. It is the predominant pentose of the hemicellulose fraction of hardwoods and agricultural residues, and its percentage is dependent on the type of lignocellulosic biomass (Abdel-Rahman et al., 2014). Several studies have showed that glucose-consuming LAB perform homolactic acid fermentation, while all xyloseconsuming LAB perform heterofermentation, producing low LA yields (≤0.676 g g−1 ) and forming by-products, such as acetic acid, formic acid and ethanol, from xylose at low concentrations (52◦ C resulted in significantly lower OD562 values, xylose consumption and LA concentration. Taken together, these data showed that optimal fermentation temperature of strain QU 50 was 50◦ C and resulted in an LA concentration of 23.7 g L−1 , LA yield of ∼1.0 g g−1 and LA productivity of 1.97 g (L·h)−1 . Time courses of l-LA production and xylose consumption by strain QU 50 under the optimal fermentation conditions are shown in Fig. 1. Notably, high LA yields (0.903–1.05 g g−1 ) were obtained at all the tested temperatures, indicating that homolactic acid fermentation occurred. Although it was not clear why LA yields exceeded 1.0 g g−1 at different temperatures, QU 50 strain may convert several
LA productivity [g (L·h)−1 ]
LA production from xylose at different pHs and temperatures
0.089 ± 0.056 0.199 ± 0.000 0.405 ± 0.000 0.645 ± 0.371 0.871 ± 0.123 2.49 ± 0.16 3.57 ± 0.16 3.94 ± 0.31 0.000
A BLAST search of the 16S rRNA gene sequence of strain QU 50 showed that it had 99.0% identity to E. faecium Aus0085 (accession number CP006620.1) and E. faecium NRRL B-2354 (accession number CP004063.1). Based on these results, strain QU 50 was identified as E. faecium QU 50.
9.07 ± 0.50 4.77 ± 0.43 23.0 ± 1.2 19.8 ± 0.4 20.0 ± 0.0 18.8 ± 0.4 13.7 ± 0.8 11.6 ± 0.9 9.00 ± 0.21
Max. LA productivity [g (L·h)−1 ]
FEMS Microbiology Letters, 2015, Vol. 362, No. 1
pH value
4
1.31 ± 0.04 2.13 ± 0.13 2.29 ± 0.42 2.44 ± 0.04 2.89 ± 0.03 2.87 ± 0.05 4.46 ± 1.15 1.04 ± 0.02 0.425
5
∗
Averages with standard deviations are based on three independent fermentations except for data presented at 55◦ C. LA, lactic acid.
0.803 ± 0.025 1.65 ± 0.03 0.865 ± 0.017 0.928 ± 0.024 1.12 ± 0.01 1.01 ± 0.01 1.97 ± 0.01 0.892 ± 0.001 0.221 0.903 ± 0.117 0.990 ± 0.020 0.925 ± 0.029 0.979 ± 0.008 1.00 ± 0.08 1.05 ± 0.13 1.02 ± 0.01 1.05 ± 0.04 1.03 1.19 ± 0.23 1.11 ± 0.49 1.39 ± 0.04 0.000 1.88 ± 0.13 1.37 ± 0.25 1.26 ± 0.01 0.000 0.000 2.34 ± 0.45 1.16 ± 0.65 2.56 ± 0.21 2.72 ± 0.01 2.44 ± 0.05 0.668 ± 0.039 0.712 ± 0.155 0.000 0.000 21.3 ± 2.1 20.0 ± 0.1 22.5 ± 1.2 22.8 ± 1.3 22.5 ± 2.2 22.9 ± 2.6 22.8 ± 0.1 20.4 ± 0.7 5.14 24 12 24 24 20 24 12 24 24 30 37 41 43 45 47 50 52 55
17.1 ± 0.1 13.6 ± 0.1 15.5 ± 0.2 16.9 ± 0.4 16.3 ± 0.2 14.6 ± 0.3 12.2 ± 0.4 5.65 ± 0.01 0.884
19.3 ± 0.1 19.8 ± 0.4 20.8 ± 0.1 22.3 ± 0.2 22.5 ± 0.2 24.1 ± 0.2 23.7 ± 0.0 21.4 ± 0.0 5.30
1.19 ± 0.21 0.645 ± 0.371 1.27 ± 0.14 1.74 ± 0.22 1.50 ± 0.12 0.749 ± 0.204 0.235 ± 0.007 0.160 ± 0.000 0.076
LA yield (g g−1 ) Ethanol (g L−1 ) Formic acid (g L−1 ) Acetic acid (g L−1 ) LA (g L−1 ) Consumed xylose (g L−1 ) Max. biomass (OD562nm ) Fermentation time (h) Temp. (◦ C)
Table 3. l-Lactic acid fermentation by E. faecium QU 50 at different temperatures with pH control of 6.5∗ .
LA productivity [g (L·h)−1 ]
Max. LA productivity [g (L·h)−1 ]
Abdel-Rahman et al.
Figure 1. Time course for fermentation performance of E. faecium QU 50 on xylose at an optimal temperature (50◦ C) and pH value (6.5). Symbols: xylose, g L−1 (closed squares); LA, g L−1 (open squares); biomass, OD562nm (closed circles); acetic acid, g L−1 (open circles), formic acid, g L−1 (closed triangles); and ethanol, g L−1 (open triangles). Fermentations were investigated in mMRS medium at xylose concentration of ca. 22 g L−1 .
components derived from fermentation medium to LA, which would result in high LA yield, as suggested previously (RomeroGarcia et al., 2009). Several xylose-consuming LAB, including Lactococcus lactis IO-1 (Tanaka et al., 2002), E. mundtii QU 25 (Abdel-Rahman et al., 2011b) and E. faecium LLAA-1 (Pessione et al., 2014) have been reported. However, low xylose concentrations (
R E S E A R C H L E T T E R – Biotechnology & Synthetic Biology
Enterococcus faecium QU 50: a novel thermophilic lactic acid bacterium for high-yield l-lactic acid production from xylose Mohamed Ali Abdel-Rahman1,2 , Yukihiro Tashiro3,4 , Takeshi Zendo1 , Kenji Sakai3 and Kenji Sonomoto1,5,∗ 1
Laboratory of Microbial Technology, Division of Systems Bioengineering, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan, 2 Botany and Microbiology Department, Faculty of Science (Boys branch), Al-Azhar University, PN:11884, Nasr City, Cairo, Egypt, 3 Laboratory of Soil Microbiology, Division of Systems Bioengineering, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, Japan, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan, 4 Institute of Advanced Study, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan and 5 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 ∗ Corresponding author: Laboratory of Microbial Technology, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan. Tel: +81-(0)92-642-3019; Fax: +81-(0)92-642-3019; E-mail: [email protected] One-sentence summary: A novel thermophilic l-lactic acid bacterium, Enterococcus faecium QU 50, was isolated. Strain QU 50 showed high lactic acid yield (∼1.0 g g−1 ) from several lignocellulose-derived sugars. QU 50 produced l-lactic acid homofermentatively (∼1.0 g g−1 yield) from low-concentration xylose. QU 50 showed efficient fermentation at 50◦ C. QU 50 is the only thermophilic LAB strain that utilizes hexose and pentose sugars homofermentatively. Editor: Michael Sauer
ABSTRACT Production of optically pure lactic acid from lignocellulosic material for commercial purposes is hampered by several difficulties, including heterofermentation of pentose sugars and high energy consumption by mesophilic lactic acid bacteria. Here, we report a novel lactic acid bacterium, strain QU 50, that has the potential to produce optically pure l-lactic acid (≥99.2%) in a homofermentative manner from xylose under thermophilic conditions. Strain QU 50 was isolated from Egyptian fertile soil and identified as Enterococcus faecium QU 50 by analyzing its sugar fermentation pattern and 16S rRNA gene sequence. Enterococcus faecium QU 50 fermented xylose efficiently to produce lactic acid over wide pH (6.0–10.0) and temperature ranges (30–52◦ C), with a pH of 6.5 and temperature of 50◦ C being optimal. To our knowledge, this is the first report of homofermentative lactic acid production from xylose by a thermophilic lactic acid bacterium. Key words: Lactate fermentation; biomass utilization; biorefinery; pentose; homofermenters; high temperature
Received: 7 November 2014; Accepted: 11 November 2014 C FEMS 2014. All rights reserved. For permissions, please e-mail: [email protected]
1
2
FEMS Microbiology Letters, 2015, Vol. 362, No. 1
INTRODUCTION Optically pure lactic acid (LA) has garnered increasing attention due to its high potential for a wide range of applications, mainly in food, chemical, cosmetic and pharmaceutical industries. Optically pure LA can only be produced by microbial fermentation because chemical synthesis produces racemic dl-LA (Tashiro et al., 2011). The main microbial LA producers are bacteria and fungi, although the latter exhibits relatively lower productivity and needs complicated requirements for LA production (Abdel-Rahman, Tashiro and Sonomoto 2013a). Bacterial producers are classified into four main groups: lactic acid bacteria (LAB), Bacillus strains, Escherichia coli and Corynebacterium glutamicum. Unlike the other bacterial producers, LAB produce LA as an anaerobic product of glycolysis with high yield and productivity. LAB are characterized as Gram-positive, non-sporing, catalase-negative, mesophilic (10–45◦ C) but aerotolerant cocci or rods that cannot use oxygen as an electron acceptor during sugar fermentation. Therefore, LAB have not included other LA producers such as Bacillus strains and some filamentous fungi (Carr, Chill and Maida 2002). LAB are comprised of about 20 genera among which the most important are Lactococcus, Enterococcus, Lactobacillus, Streptococcus, Leuconostoc, Pediococcus, Carnobacterium, Oenococcus, Aerococcus, Tetragenococcus, Weissella and Vagococcus (Reddy et al., 2008; Abdel-Rahman, Tashiro and Sonomoto 2013a). Currently, lignocellulosic biomasses are considered as an economically attractive feedstock for LA production, which does not significantly affect food supply and price. Lignocellulose is composed of cellulose (35–50%), hemicellulose (20– 40%) and lignin (10–30%); thus, it contains large amounts of fermentable sugars in the form of glucose and xylose (AbdelRahman, Tashiro and Sonomoto 2011a, 2013a). Xylose is the second most abundant sugar in nature, next to glucose. It is the predominant pentose of the hemicellulose fraction of hardwoods and agricultural residues, and its percentage is dependent on the type of lignocellulosic biomass (Abdel-Rahman et al., 2014). Several studies have showed that glucose-consuming LAB perform homolactic acid fermentation, while all xyloseconsuming LAB perform heterofermentation, producing low LA yields (≤0.676 g g−1 ) and forming by-products, such as acetic acid, formic acid and ethanol, from xylose at low concentrations (52◦ C resulted in significantly lower OD562 values, xylose consumption and LA concentration. Taken together, these data showed that optimal fermentation temperature of strain QU 50 was 50◦ C and resulted in an LA concentration of 23.7 g L−1 , LA yield of ∼1.0 g g−1 and LA productivity of 1.97 g (L·h)−1 . Time courses of l-LA production and xylose consumption by strain QU 50 under the optimal fermentation conditions are shown in Fig. 1. Notably, high LA yields (0.903–1.05 g g−1 ) were obtained at all the tested temperatures, indicating that homolactic acid fermentation occurred. Although it was not clear why LA yields exceeded 1.0 g g−1 at different temperatures, QU 50 strain may convert several
LA productivity [g (L·h)−1 ]
LA production from xylose at different pHs and temperatures
0.089 ± 0.056 0.199 ± 0.000 0.405 ± 0.000 0.645 ± 0.371 0.871 ± 0.123 2.49 ± 0.16 3.57 ± 0.16 3.94 ± 0.31 0.000
A BLAST search of the 16S rRNA gene sequence of strain QU 50 showed that it had 99.0% identity to E. faecium Aus0085 (accession number CP006620.1) and E. faecium NRRL B-2354 (accession number CP004063.1). Based on these results, strain QU 50 was identified as E. faecium QU 50.
9.07 ± 0.50 4.77 ± 0.43 23.0 ± 1.2 19.8 ± 0.4 20.0 ± 0.0 18.8 ± 0.4 13.7 ± 0.8 11.6 ± 0.9 9.00 ± 0.21
Max. LA productivity [g (L·h)−1 ]
FEMS Microbiology Letters, 2015, Vol. 362, No. 1
pH value
4
1.31 ± 0.04 2.13 ± 0.13 2.29 ± 0.42 2.44 ± 0.04 2.89 ± 0.03 2.87 ± 0.05 4.46 ± 1.15 1.04 ± 0.02 0.425
5
∗
Averages with standard deviations are based on three independent fermentations except for data presented at 55◦ C. LA, lactic acid.
0.803 ± 0.025 1.65 ± 0.03 0.865 ± 0.017 0.928 ± 0.024 1.12 ± 0.01 1.01 ± 0.01 1.97 ± 0.01 0.892 ± 0.001 0.221 0.903 ± 0.117 0.990 ± 0.020 0.925 ± 0.029 0.979 ± 0.008 1.00 ± 0.08 1.05 ± 0.13 1.02 ± 0.01 1.05 ± 0.04 1.03 1.19 ± 0.23 1.11 ± 0.49 1.39 ± 0.04 0.000 1.88 ± 0.13 1.37 ± 0.25 1.26 ± 0.01 0.000 0.000 2.34 ± 0.45 1.16 ± 0.65 2.56 ± 0.21 2.72 ± 0.01 2.44 ± 0.05 0.668 ± 0.039 0.712 ± 0.155 0.000 0.000 21.3 ± 2.1 20.0 ± 0.1 22.5 ± 1.2 22.8 ± 1.3 22.5 ± 2.2 22.9 ± 2.6 22.8 ± 0.1 20.4 ± 0.7 5.14 24 12 24 24 20 24 12 24 24 30 37 41 43 45 47 50 52 55
17.1 ± 0.1 13.6 ± 0.1 15.5 ± 0.2 16.9 ± 0.4 16.3 ± 0.2 14.6 ± 0.3 12.2 ± 0.4 5.65 ± 0.01 0.884
19.3 ± 0.1 19.8 ± 0.4 20.8 ± 0.1 22.3 ± 0.2 22.5 ± 0.2 24.1 ± 0.2 23.7 ± 0.0 21.4 ± 0.0 5.30
1.19 ± 0.21 0.645 ± 0.371 1.27 ± 0.14 1.74 ± 0.22 1.50 ± 0.12 0.749 ± 0.204 0.235 ± 0.007 0.160 ± 0.000 0.076
LA yield (g g−1 ) Ethanol (g L−1 ) Formic acid (g L−1 ) Acetic acid (g L−1 ) LA (g L−1 ) Consumed xylose (g L−1 ) Max. biomass (OD562nm ) Fermentation time (h) Temp. (◦ C)
Table 3. l-Lactic acid fermentation by E. faecium QU 50 at different temperatures with pH control of 6.5∗ .
LA productivity [g (L·h)−1 ]
Max. LA productivity [g (L·h)−1 ]
Abdel-Rahman et al.
Figure 1. Time course for fermentation performance of E. faecium QU 50 on xylose at an optimal temperature (50◦ C) and pH value (6.5). Symbols: xylose, g L−1 (closed squares); LA, g L−1 (open squares); biomass, OD562nm (closed circles); acetic acid, g L−1 (open circles), formic acid, g L−1 (closed triangles); and ethanol, g L−1 (open triangles). Fermentations were investigated in mMRS medium at xylose concentration of ca. 22 g L−1 .
components derived from fermentation medium to LA, which would result in high LA yield, as suggested previously (RomeroGarcia et al., 2009). Several xylose-consuming LAB, including Lactococcus lactis IO-1 (Tanaka et al., 2002), E. mundtii QU 25 (Abdel-Rahman et al., 2011b) and E. faecium LLAA-1 (Pessione et al., 2014) have been reported. However, low xylose concentrations (
