New application of Bacillus strains for optically pure l-lactic acid production: general overview and future prospects

Figures & data

Table 1. Special features of LA-producing Bacillus strains and comparison to other LA producers.

Characterization	Lactic acid producers
	Bacillus strains	Lactic acid bacteria (LAB)	Rhizopus sp.
Nutritional requirements for growth	Requirement of less nutrients to grow than LAB Ability to grow in mineral salt medium containing low-organic nitrogen sources (e.g. yeast extract)	Requirement of nutritionally rich medium (amino acids, vitamins, and nucleotides)	Requirement of less nutrients to grow than LAB Ability to grow in simple inorganic salt containing media
Tolerance to high temperature	Optimum temperature for LA production: 45–60 °C Maximum temperature for cell growth: 70 °C with B. coagulans NBRC 12583	Optimum temperature for LA production: 30–43 °C with general LAB, 50 °C with E. faecium QU50 Maximum temperature for cell growth: 55 °C with E. faecium QU50	Optimum temperature for LA production: 27–35 °C Maximum temperature for cell growth: 45 °C with Rhizopus oligosporus TISTR 3518
Tolerance to pH	Optimum pH for LA production: 5.0–9.0 Sensitive to acidic pH (<5.0) and alkaline pH (>9.0) except for tolerant to pH at 10 with Bacillus sp. WL-S20	Optimum pH for LA production: 4.0–7.0 Tolerant to low pH ~4.0 (Lb. plantarum ATCC 21028) and to high pH ~10.5 (Alkalibacterium olivoapovliticus)	Optimum pH for LA production: 5.0–6.0 Tolerant to low pH ~3.5 (R. oryzae ATCC 52311)
Ability to produce optically pure LA	l-LA producers (Major strains): 97–100% of optical purity d-LA producer (B. laevolacticus NCIB 10269): >99% of optical purity	l-LA producers (genera of Lactococcus, some Lactobacillus, Enterococcus, etc.) : >~95% d-LA producers (genus Leuconostoc, Lactobacillus delbrueckii subsp. lactis, etc.) : >~99%	l-LA producers (Major strains): 98.5–100% of optical purity l-LA
Sugar metabolism	Hexoses: ferment to LA and by-products (acetic acid, formic acid, 2,3-butanediol) via EMP (max. LA yield >0.90 g/g) Pentoses: ferment to LA via PPP and EMP (max. LA yield >0.90 g/g)	Hexoses: ferment to LA and by-products (acetic acid, formic acid, ethanol) via EMP (max. LA yield >0.90 g/g) Pentoses: ferment to LA via PPP (max. LA yield >0.90 g/g) and PKP (max. LA yield < ca. 0.60 g/g)	Hexoses: ferment to LA and by-products (ethanol, CO2, fumaric acid, malic acid, citric acid,) via EMP and tricarboxylic acid pathway (max. LA yield ≈ 0.71–0.79 g/g) Pentoses: ferment to via PPP (max. LA yield ≈ 054–0.65 g/g)
Oxygen demand	Facultatively anaerobes In the absence of O2: grow	Facultatively anaerobes In the presence of O2: grow and tolerant to O2, but cannot utilize O2 as the electron acceptor in metabolism In the absence of O2: grow very well	Obligatory aerobes In the presence of O2: grow very well using O2 as the electron acceptor in metabolism In the absence of O2: cannot grow

Fig. 1. Metabolic pathway for LA production from several sugars (xylose, glucose, arabinose), cellulose, and starch derived from various biomasses via homofermentative (EMP/PPP) (A) and heterofermentative (PK) (B) pathways.

Note: Enzymes: (a) hexokinase; (b) glucose 6P isomerase; (c) 6-phosphofructo kinase; (d) triose phosphate isomerase; (e) lactate dehydrogenase; (f) xylose isomerase; (g) xylulose kinase; (h) epimerase; (i) transaldolase; (j) transketolase; (k) glucose 6P dehydrogenase; (l) phosphoketolase.

Table 2. Lactic acid production efficiencies among the Bacillus strains under batch and repeated batch fermentation modes.

Fermentation substrate	Strains	Lactic acid				Fermentation parameters	Fermentation modes	References
		C (g/L)	Y (g/g)	P (g/L h)	OP (%)^†
Glucose	B. thermoamylovorans CNCM I-1378^T	11.8	nd	ca. 0.49	100	pH control at 7.0; Ferm.temp., 50 °C; Yeast extract, 20 g/L	Batch	^89^)
Molasses	B. coagulans TB/04	55.0	0.93	nd	99	pH control at 6.4; Ferm.temp., 52 °C; Yeast extract, 2 g/L	Batch	^83^)
Soft wood hydrolysate	B. coagulans BS121	13.7	nd	ca. 0.19	99	pH control at 6.0; Ferm.temp., 60 °C; Yeast extract, 4 g/L	Batch	^69^)
Acid hydrolysis sugarcane baggase	B. coagulans 17C5	55.8	ca. 0.93	ca. 0.80	99.5	pH control at 5.0; Ferm.temp., 50 °C; Corn steep liquor, 5 g/L	Batch	^60^)
Acid hydrolysis of sugarcane	B. coagulans 36D1	36.0	nd	nd	99.0	pH control at 5.0; Ferm.temp., 50 °C	Batch	^59^)
Saccharified food waste	B. coagulans NBRC 12583^T	86.0	0.97	0.72	97.0	pH control at 6.5; Ferm.temp., 55 °C	Batch	^18^)
Saccharified food waste	B. licheniformis TY7	40.0	1.35	2.50	97.0	pH control at 6.5; Ferm.temp., 55 °C	Batch	^90^)
Paper sludge hydrolysates	B. coagulans 36D1	92.8	0.77	0.96	99.5	pH control at 5.0; Ferm.temp., 50; Corn steep liquor, 5 g/L	Batch	^72^)
Paper sludge hydrolysate	B. coagulans P4–102B	91.78	0.78	0.82	99.5	pH control at 5.0; Ferm.temp., 50 °C; Corn steep liquor, 5 g/L	Batch	^72^)
Corn fiber hydrolysate	B. coagulans MXL-9	40.2	0.58	0.54	99.5	pH control at 6.0; Ferm.temp., 50 °C; Yeast extract, 10 g/L	Batch	^73^)
Hot water-extracted Siberian larch	B. coagulans MXL-9	33.0	0.94	0.55	99	pH control at 7.0; Ferm.temp., 50 °C; Yeast extract, 5 g/L; Tryptone, 10 g/L	Batch	^52^)
Glucose	B. coagulans 2–6	63	0.94	1.4	99.4	pH control at 6.0; Ferm.temp., 50 °C; Yeast extract, 10 g/L	Batch	^51^)
Glucose	B. coagulans 2–6	107.0	0.95	2.90	99.8	pH control at 6.0; Ferm.temp., 50 °C; Yeast extract, 10 g/L	Repeated batch	^51^)
Cellobiose	B. licheniformis BL1	4.4	0.86	0.30	>99.0	pH control at 7.0; Ferm.temp., 50 °C; Yeast extract, 5 g/L	Batch	^34^)
Corn stover hydrolyzate	B. coagulans XZL4	81.0	0.98	1.86	99.5	pH control at 6.0; Ferm.temp., 50 °C	Batch	^91^)
Glucose	B. subtilis MUR1	143.2	0.90	2.75	99.5	pH control at 7.5; Ferm. temp., 37 °C; Air flow rate, 50 vvm; Yeast extract, 75 g/L	Batch	^36^)
Corn stover hydrolysate/Xylose	B. coagulans NL01	75.0	0.75	1.04	99.8	pH control at 6.5; Ferm.temp., 50 °C; Yeast extract, 2.5 g/L	Batch	^92^)
Oil palm empty fruit bunch hydrolysate	B. coagulans JI12	59.2	0.97	6.2	99.6	pH control at 6.0; Ferm.temp., 50 °C; Yeast extract, 10 g/L	Batch	^75^)
Xylose	B. coagulans C106	83.6	0.98	7.5	99.6	pH control at 6.0; Ferm.temp., 50 °C; Yeast extract, 20 g/L	Batch	^42^)
		140.9	0.98	4.8			Batch
Glucose	B. coagulans WCP10–4	210.0	0.99	3.50	100	pH control at 6.0; Ferm.temp., 50 °C; Yeast extract, 20 g/L	Batch	^87^)
Oil palm empty fruit bunch hydrolysate	B. coagulans JI12	80.6	ca. 1.19	3.40	99.6	pH control at 6.0; Ferm.temp., 50 °C; Yeast extract, 10 g/L	Batch	^76^)
Glucose^a/Food waste	B. coagulans M21	12.5/35.1	0.62/0.86	0.31/0.58	100/100	pH control at 7.0; Ferm.temp., 50 °C; Yeast extract, 5 g/L	Batch	^50^)
Wheat straw hydrolysate	B. coagulans IPE22	56.4	0.96	2.35	100	pH control at 6.0; Ferm.temp., 52 °C; Yeast extract, 4 g/L	Repeated batch	^79^)
Soluble starch	Bacillus sp. MC-07	16.6	0.977	0.70	100	pH control at 7.0; Ferm. temp., 50 °C; Yeast extract, 0.01 g/L	Batch	^33^)

Notes:

C, concentration; Y, yield; P, productivity; nd, not described. The optical purity (%) of l-LA was defined as ([L]−[D]) × 100/([L]+[D]), where [L] and [D] denote the concentrations of l-LA and d-LA, respectively.

^† OP, optical purity (only l-isomer)

^a Under aerobic condition (K_La = 1.43 min⁻¹ see Ref.⁴⁸⁾

Table 3. Lactic acid production in fed-batch and continuous fermentation processes.

Fermentation substrate	Strains	Lactic acid				Fermentation mode	Fermentation parameters	References
		C (g/L)	Y (g/g)	P (g/L h)	OP (%)
Glucose	Bacillus laevolacticus NCIB 10269	45.0	1.0	ca 13.1	D-isomer, >99	Continuous	pH control at 6.0; Dilution rate, 0.07 h^−1; Ferm. temp., 30 °C; Yeast extract, 2 g/L; Anaerobic condition by sparging N2 gas	^17^)
Sucrose	B. coagulans TB/04	20.1	0.93	6.1	>99	Continuous	pH control at 6.4; Dilution rate, 0.03 h^−1; Ferm.temp., 50 °C, Yeast extract, 4 g/L	^83^)
Glucose	B. subtilis MUR1	55.9	ca. 0.95	16.8	99.5	Continuous	pH control at 7.5; Dilution rate, 0.3 h^−1; Ferm.temp., 37 °C; Agitation speed, 50 rpm; Air flow rate, 3 vvm	^35^)
Glucose	B. subtilis MUR1	183.5	0.98	3.52	99.5	Fed-batch with exponential feeding	pH control at 7.5; Ferm.temp., 37 °C; Air flow rate, 50 vvm; Yeast extract, 75 g/L	^36^)
Lime treated wheat straw	B. coagulans DSM2314	40.7	0.43	0.74	97.2	Fed-batch with constant feeding	pH control at 6.0; Ferm.temp., 50 °C; Yeast extract, 10 g/L	^72^)
Corncob molasses	B. coagulans XZL9	74.7	0.50	0.37	99.5	Fed-batch with pulse feeding	pH control at 6.0; Ferm.temp., 50 °C; Yeast extract, 10 g/L	^74^)
Cellulose	B. coagulans 36D1	80.0	0.88	ca. 0.84	99.0	Fed-batch with constant feeding	pH control at 5.0; Ferm.temp., 50 °C; Corn steep liquor, 7.5 g/L	^43^)
Glucose	Bacillus sp. WL-S20	225.0	0.99	1.04	100	Fed-batch with pulse feeding	pH controlled at 9.0; Ferm.temp., 45 °C; Peanut meal, 20 g/L	^37^)
Xylose	B. coagulans C106	215.7	0.95	4.0	99.6	Fed-batch with pulse feeding	pH control at 6.0; Ferm.temp., 50 °C; Yeast extract, 20 g/L	^42^)
Corn stover hydrolysate	B. coagulans NL01	75.0	0.75	1.04	>99	Fed-batch with pulse feeding	pH controlled at 6.5; Ferm.temp., 50 °C; Yeast extract, 2.5 g/L	^92^)
Acid treated corn stover hydrolysate	B. coagulans P38	180.0	0.98	2.40	100	Fed-batch with pulse feeding	pH controlled at 4.8–5.0; Ferm.temp., 42 °C	^50^)
Jeruselem antichoke hydrolysate	B. coagulans XZL4	134.0	0.96	2.50	>99.0	Fed-batch with pulse feeding	pH controlled at 5.2–5.5; Ferm.temp., 50 °C; Corn steep powder, 10 g/L	^76^)
Glucose	B. coagulans 2–6	122.0	nd	2.50	99.2	Fed-batch with exponential feeding	pH controlled at 5.6; Ferm.temp., 50 °C for 36 h and increased up to 55 °C at 0.2 °C/h; Yeast extract, 5 g/L	^93^)
Mixture of untreated cane molasses and glucose	B. coagulans H-1	168.3	0.88	2.10	>99	Fed-batch with constant feeding	pH controlled at 6.2; Ferm.temp., 52 °C; Yeast extract, 10 g/L	^94^)

Note: C, concentration; Y, yield; P, productivity; OP, optical purity (only l-isomer unless indicated); nd, not described.

Combet-Blanc Y, Ollivier B, Streicher C, et al. Bacillus thermoamylovorans sp. nov., a moderately thermophilic and amylolytic bacterium. Int. J. Syst. Bacteriol. 1995;45:9–16.10.1099/00207713-45-1-9

 PubMedGoogle Scholar

Payot T, Chemaly Z, Fick M. Lactic acid production by Bacillus coagulans—kinetic studies and optimization of culture medium for batch and continuous fermentations. Enzyme Microb. Technol. 1999;24:191–199.10.1016/S0141-0229(98)00098-2

 Web of Science ®Google Scholar

Neureiter M, Danner H, Madzingaidzo L, et al. Lignocellulose feedstocks for the production of lactic acid. Chem. Biochem. Eng. Q. 2004;18:55–63.

 Web of Science ®Google Scholar

Patel M, Ou M, Ingram LO, et al. Fermentation of sugar cane bagasse hemicellulose hydrolysate to l(+)-lactic acid by a thermotolerant acidophilic Bacillus sp. Biotechnol. Lett. 2004;26:865–868.10.1023/B:bile.0000025893.27700.5c

 Google Scholar

Patel MA, Ou MS, Harbrucker R, et al. Isolation and characterization of acid-tolerant, thermophilic bacteria for effective fermentation of biomass-derived sugars to lactic acid. Appl. Environ. Microbiol. 2006;72:3228–3235.10.1128/AEM.72.5.3228-3235.2006

 PubMed Web of Science ®Google Scholar

Sakai K, Ezaki Y. Open l-lactic acid fermentation of food refuse using thermophilic Bacillus coagulans and fluorescence in situ hybridization analysis of microflora. J. Biosci. Bioeng. 2006;101:457–463.10.1263/jbb.101.457

 PubMed Web of Science ®Google Scholar

Sakai K, Yamanami T. Thermotolerant Bacillus licheniformis TY7 produces optically active l-lactic acid from kitchen refuse under open condition. J. Biosci. Bioeng. 2006;102:132–134.10.1263/jbb.102.132

 Google Scholar

Budhavaram NK, Fan Z. Production of lactic acid from paper sludge using acid-tolerant, thermophilic Bacillus coagulan strains. Bioresour. Technol. 2009;100:5966–5972.10.1016/j.biortech.2009.01.080

 PubMed Web of Science ®Google Scholar

Bischoff KM, Liu S, Hughes SR, et al. Fermentation of corn fiber hydrolysate to lactic acid by the moderate thermophile Bacillus coagulans. Biotechnol. Lett. 2010;32:823–828.10.1007/s10529-010-0222-z

 PubMed Web of Science ®Google Scholar

Walton SL, Bischoff KM, van Heiningen ARP, et al. Production of lactic acid from hemicellulose extracts by Bacillus coagulans MXL-9. J. Ind. Microbiol. Biotechnol. 2010;37:823–830.10.1007/s10295-010-0727-4

 PubMed Web of Science ®Google Scholar

Zhao B, Wang L, Ma C. Repeated open fermentative production of optically pure l-lactic acid using a thermophilic Bacillus sp. strain. Bioresour. Technol. 2010;101:6494–6498.10.1016/j.biortech.2010.03.051

 PubMed Web of Science ®Google Scholar

Wang Q, Zhao X, Chamu J, et al. Isolation, characterization and evolution of a new thermophilic Bacillus licheniformis for lactic acid production in mineral salts medium. Bioresour. Technol. 2011;102:8152–8158.10.1016/j.biortech.2011.06.003

 PubMed Web of Science ®Google Scholar

Xue Z, Wang L, Ju J, et al. Efficient production of polymer-grade l-lactic acid from corn stover hydrolyzate by thermophilic Bacillus sp. strain XZL4. SpringerPlus. 2012;1:1–7.

 PubMed Web of Science ®Google Scholar

Gao T, Wong Y, Ng C, et al. l-Lactic acid production by Bacillus subtilis MUR1. Bioresour. Technol. 2012;121:105–110.10.1016/j.biortech.2012.06.108

 PubMed Web of Science ®Google Scholar

Ouyang J, Ma R, Zheng Z, et al. Open fermentative production of l-lactic acid by Bacillus sp. strain NL01 using lignocellulosic hydrolyzates as low-cost raw material. Bioresour. Technol. 2013;135:475–480.10.1016/j.biortech.2012.09.096

 PubMed Web of Science ®Google Scholar

Ye L, Bin Hudari MS, Zhi L, et al. Simultaneous detoxification, saccharification and co-fermentation of oil palm empty fruit bunch hydrolysate for l-lactic acid production by Bacillus coagulans JI12. Biochem. Eng. J. 2013;83:16–21.

 Web of Science ®Google Scholar

Ye L, Zhou X, Bin Hudari MS, et al. Highly efficient production of l-lactic acid from xylose by newly isolated Bacillus coagulans C106. Bioresour. Technol. 2013;132:38–44.10.1016/j.biortech.2013.01.011

 PubMed Web of Science ®Google Scholar

Zhou X, Ye L, Wu JC. Efficient production of l-lactic acid by newly isolated thermophilic Bacillus coagulans WCP10-4 with high glucose tolerance. Appl. Microbiol. Biotechnol. 2013;97:4309–4314.10.1007/s00253-013-4710-7

 PubMed Web of Science ®Google Scholar

Ye L, Bin Hudari MS, Zhou X, et al. Conversion of acid hydrolysate of oil palm empty fruit bunch to l-lactic acid by newly isolated Bacillus coagulans JI12. Appl. Microbiol. Biotechnol. 2013;97:4831–4838.10.1007/s00253-013-4788-y

 PubMed Web of Science ®Google Scholar

Peng L, Wang L, Che C, et al. Bacillus sp. strain P38: an efficient producer of l-lactate from cellulosic hydrolysate, with high tolerance for 2-furfural. Bioresour. Technol. 2013;149:169–176.10.1016/j.biortech.2013.09.047

 Google Scholar

Zhang Y, Chen X, Qi B, et al. Improving lactic acid productivity from wheat straw hydrolysates by membrane integrated repeated batch fermentation under non-sterilized conditions. Bioresour. Technol. 2014;163:160–166.

 PubMed Web of Science ®Google Scholar

Poudel P, Tashiro Y, Miyamoto H, et al. Direct starch fermentation to l-lactic acid by a newly isolated thermophilic strain, Bacillus sp. MC-07. J. Ind. Microbiol. Biotechnol. 2015;42:143–149.10.1007/s10295-014-1534-0

 Google Scholar

Albert RA, Archambault J, Rosselló-Mora R, et al. Bacillus acidicola sp. nov., a novel mesophilic, acidophilic species isolated from acidic Sphagnum peat bogs in Wisconsin. Int. J. Syst. Evol. Microbiol. 2005;55:2125–2130.10.1099/ijs.0.02337-0

 PubMed Web of Science ®Google Scholar

De Boer JP, de Mattos MJT, Neijssel OM. d(−)Lactic acid production by suspended and aggregated continuous cultures of Bacillus laevolacticus. Appl. Microbiol. Biotechnol. 1990;34:149–153.10.1007/BF00166771

 Google Scholar

Gao T, Ho KP. l-Lactic acid production by Bacillus subtilis MUR1 in continuous culture. J. Biotechnol. 2013;168:646–651.10.1016/j.jbiotec.2013.09.023

 PubMed Web of Science ®Google Scholar

Wang L, Zhao B, Liu B, et al. Efficient production of l-lactic acid from corncob molasses, a waste by-product in xylitol production, by a newly isolated xylose utilizing Bacillus sp. strain. Bioresour. Technol. 2010;101:7908–7915.10.1016/j.biortech.2010.05.031

 PubMed Web of Science ®Google Scholar

Ramos HC, Hoffmann T, Marino M, et al. Fermentative metabolism of Bacillus subtilis: physiology and regulation of gene expression. J. Bacteriol. 2000;182:3072–3080.10.1128/JB.182.11.3072-3080.2000

 PubMed Web of Science ®Google Scholar

Meng Y, Xue Y, Yu B, et al. Efficient production of l-lactic acid with high optical purity by alkaliphilic Bacillus sp. WL-S20. Bioresour. Technol. 2012;116:334–339.10.1016/j.biortech.2012.03.103

 PubMed Web of Science ®Google Scholar

Qin J, Zhao B, Wang X, et al. Non-sterilized fermentative production of polymer-grade l-lactic acid by a newly isolated thermophilic strain Bacillus sp. 2–6. PLoS One. 2009;4:e4359.

 PubMed Web of Science ®Google Scholar

Xu K, Xu P. Efficient production of l-lactic acid using co-feeding strategy based on cane molasses/glucose carbon sources. Bioresour. Technol. 2013;153:23–29.

 PubMed Web of Science ®Google Scholar