RESEARCH PAPER
New Biotechnology Volume 31, Number 3 May 2014
Research Paper
Ethanol production from glycerolcontaining biodiesel waste by Klebsiella variicola shows maximum productivity under alkaline conditions Toshihiro Suzuki1, Chiaki Nishikawa1, Kohei Seta1, Toshiya Shigeno2 and Toshiaki Nakajima-Kambe1 1 2
Faculty of Life and Environmental Sciences (Bioindustrial Sciences), University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan Tsukuba Environmental Microorganism Institute, 8-1 Sakuragaoka, Tsukuba, Ibaraki 300-1271, Japan
Biodiesel fuel (BDF) waste contains large amounts of crude glycerol as a by-product, and has a high alkaline pH. With regard to microbial conversion of ethanol from BDF-derived glycerol, bacteria that can produce ethanol at alkaline pH have not been reported to date. Isolation of bacteria that shows maximum productivity under alkaline conditions is essential to effective production of ethanol from BDF-derived glycerol. In this study, we isolated the Klebsiella variicola TB-83 strain, which demonstrated maximum ethanol productivity at alkaline pH. Strain TB-83 showed effective usage of crude glycerol with maximum ethanol production at pH 8.0–9.0, and the culture pH was finally neutralized by formate, a by-product. In addition, the ethanol productivity of strain TB-83 under various culture conditions was investigated. Ethanol production was more efficient with the addition of yeast extract. Strain TB-83 produced 9.8 g/L ethanol (0.86 mol/mol glycerol) from cooking oil-derived BDF waste. Ethanol production from cooking oil-derived BDF waste was higher than that of new frying oil-derived BDF and pure-glycerol. This is the first report to demonstrate that the K. variicola strain TB-83 has the ability to produce ethanol from glycerol at alkaline pH.
Introduction Biodiesel fuel (BDF) is biomass fuel that is used as an alternative fuel of diesel oil. BDF, a carbon neutral fuel, is also made from vegetable oils and animal fats [1–4]. Almost all BDF production is carried out by transesterification with methanol in the presence of an alkaline catalyst [3,5]. However, BDF waste has a high alkaline pH, and contains a large amount of crude glycerol as a by-product [6]. Since almost all BDF-derived glycerol is waste oil-derived, it has no applied use, and is almost always incinerated. Therefore, the treatment of BDF-derived crude glycerol is an environmental problem. Bacteria that can utilize glycerol as a sole carbon source are known to produce some valuable metabolites [3,7]. Recent studies have reported that the major metabolites derived from glycerol-assimilation are 1,3-propanediol (1,3-PD) [8–11], hydrogen [12–14], and
Corresponding author: Nakajima-Kambe, T. ([email protected]) www.elsevier.com/locate/nbt
246
ethanol [13,15,16]. Bio-ethanol is particularly valuable as its biomass energy as well as BDF. The production of bio-ethanol is an efficient use of waste and decreases burdens on the environment. In the fermentation of glycerol, the initial culture pH is neutral but gradually decreases through the accumulation of organic acid by-products [12–17]. The same problem of glycerol residue treatment also emerges in the case of BDF. In addition, when pure glycerol is used, many ethanol producers show high ethanol production, whereas ethanol productivity decreases using BDF waste [13,18]. Recent studies of BDF waste have revealed that the family Enterobacteriaceae and its mutant strains produce higher amounts of ethanol when using BDF than pure glycerol. Klebsiella pneumoniae GEM167, a gamma-irradiation mutant, produces 25.0 g/L of ethanol with a productivity rate of 0.93 g L1 hour1 from BDF waste [19]. Saisaard et al. reported that Enterobacter aerogenes TISTR1468 produced 24.5 g/L using waste
http://dx.doi.org/10.1016/j.nbt.2014.03.005 1871-6784/ß 2014 Elsevier B.V. All rights reserved.
New Biotechnology Volume 31, Number 3 May 2014
Materials and methods Media and reagents Screening medium containing 10.0 g KH2PO4, 1.0 g NH4Cl, 0.50 g yeast extract (Difco; Detroit, MI, USA), and 100.0 g glycerol in 1 L of distilled water (adjusted to pH 7.0 with 6 N NaOH) was used for screening bacteria. Glycerol basal medium, containing 10.0 g KH2PO4, 1.0 g NH4Cl, 0.50 g yeast extract (Difco) 1.0 g casamino acid (Difco), and 20.0 g glycerol in 1 L of distilled water, was used for isolating and culturing bacteria. When examining BDF waste, glycerol was replaced with BDF waste. If not otherwise specified, all reagents for this study were obtained from Wako pure chemical industries, Ltd (Osaka, Japan). Two types of BDF waste were provided: BDF waste containing white-colored ash and very little impurities (Niigata Institute of Technology), which is a new frying oil-derived BDF, and brown BDF waste containing a lot of ash and impurities such as fats, oils, and triglycerides, which is a waste cooking oil-derived BDF (Nagasaki Prefectural Institute for Environmental Research and Public Health). Total organic carbon (TOC) containing the two types of BDF waste were analyzed with Shimadzu TOC-V, a total organic carbon analyzer (Shimadzu, Kyoto, Japan), and glycerol concentration was measured with high-performance liquid chromatography (HPLC). To examine the fatty acids composition, BDF waste (20 mg) was methyl-esterified with 5% hydrogen chloride methanol solution (Wako) at 958C for 3 hours, and then extracted with n-hexane. The n-hexane layer was analyzed by GC/MS, TRACE GC gas chromatography and FINNIGAN POLARIS Q mass spectrometer (Thermo Scientific, Tokyo, Japan) equipped with a DB-1MS fused Silica Capillary column (30 m 0.25 mm 0.25 mm) (Agilent Technologies, Inc., Fort Collins, CO, USA), and helium was used as career gas. Analysis was performed according to the method of Ban et al. [22].
Sampling and isolation of bacteria Soil samples (500 mg) from Tsukuba, Japan were collected in test tubes (f25 mm 200 mm) and cultured at 258C in 30 mL of the screening medium inserted into a Durham fermentation tube (f9 mm 30 mm). After bubbles were observed in the Durham fermentation tube, 0.5 mL of the broth was inoculated into the same medium. This isolation procedure was repeated at least three times. A portion of the broth was streaked onto glycerol basal agar medium and cultured at 308C for 7 days. Determination of bio-
chemical characteristic was performed by TechnoSuruga Laboratory Co., Ltd. (Shizuoka, Japan).
Genetic manipulations Total DNA isolation from Klebsiella variicola TB-83 was conducted as described below. Cells were harvested from 5 mL nutrient broth (NB) (Difco) liquid medium by centrifugation and washed twice with 567 mL TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0). Cells were re-suspended in same volume of TE buffer to which 30 mL 10% sodium dodecyl sulfate and 3 mL Proteinase K solution (20 mg/mL in TE) were added followed by incubation at 378C for 1 hour. NaCl (2 mL; 5 M) was added followed by mixing for 30 min, and then 80 mL hexadecyltrimethyl ammonium bromide (CTAB)/NaCl solution was added to the cell suspension. The mixture was incubated at 658C for 10 min, and then chloroform:isoamyl alcohol (24:1) was added followed by shaking. The mixture was centrifuged at 7000 g for 5 min and then the supernatant was isopropanol-precipitated. The precipitate was dissolved in TE buffer, extracted with phenol, incubated with RNase (100 mg/mL) at 378C for 1 hour, and extracted again with phenol:chloroform:isoamyl alcohol (25:24:1). DNA manipulations of Escherichia coli were performed according to Sambrook et al. [23]. The 16S rRNA gene coding sequence was amplified from total DNA as a template using primers 27F (50 -AGAGTTTGATCCTGGCTCAG-30 ) and 1492R (50 -TGACTGACTGAGGYTACCTTGTTACGACTT-30 ) [24]. Polymerase chain reaction (PCR) amplification was performed with the MJ Mini Personal Thermal Cycler (Bio-Rad; Hercules, CA) using Ex Taq DNA polymerase (Takara; Kyoto, Japan). PCR was conducted as follows: 948C for 2 min, followed by 30 cycles of 988C for 15 s, 608C for 20 s, and 748C for 1 min. The PCR product was ligated into the pGEM-T Easy vector (Promega; Madison, WI, USA) for sequence analysis, and then transformed in E. coli DH10B as a host strain. DNA sequencing was carried out by cycle sequencing using the BigDye terminator v3.1 cycle sequencing kit (Applied Biosystems; Foster City, CA, USA) and analyzed with an ABI PRISMTM 3130NT genetic analyzer (Applied Biosystems).
Phylogenetic analysis Homology searches were conducted using the BLAST program (http://www.ncbi.nlm.nih.gov/BLAST/). The sequences were aligned using the CLUSTALW ver. 1.83 program. Phylogenetic trees were constructed using TreeViewX software by the neighborjoining method. A bootstrap analysis with 100 trial replications was performed to determine the reliability of clustering patterns.
Culture conditions for ethanol production Ethanol production was performed using glycerol basal medium under various pH, temperature, and culture conditions. One loop of strain TB-83 grown on an NB agar plate was suspended with 1 mL of glycerol basal liquid medium. The suspension was inoculated into a test tube (f25 mm 200 mm) containing 30 mL of glycerol basal medium (final OD580 = 0.01) and incubated for various periods at 258C under static culture. One milliliter of culture was sequentially collected and analyzed for OD580, culture pH, and product concentrations. To examine batch cultures in basal medium using BDF waste, glycerol was replaced with BDF waste diluted 20 times with distilled water. www.elsevier.com/locate/nbt
247
Research Paper
cooking oil-derived BDF [20]. Recently, Kluyvera cryocrescens S26, a wild strain, was shown to produce 27 g/L with 80% yield by batch culture of palm oil-derived crude glycerol [21]. However, bacteria that can produce ethanol at alkaline pH have not been reported. In addition to the identification of bacteria that can produce ethanol at alkaline pH, evaluation of ethanol productivity is essential for high-efficiency ethanol production using BDF waste from the standpoint of production costs. To overcome the problem of ethanol production at alkaline pH, we isolated glycerol-assimilating bacteria from a soil environment and investigated their ethanol productivities at alkaline pH under various culture conditions. In addition, effects of the culture components in ethanol production were examined.
RESEARCH PAPER
RESEARCH PAPER
Measurement of metabolite concentrations
Research Paper
The supernatant of 1 mL culture was filtered immediately through a 0.2-mm polyvinylidene fluoride single-step filter vial (Thomson Instrument Company; Oceanside, CA, USA). Ethanol and other metabolite concentrations were measured by Waters alliance 2695 high-performance liquid chromatography (HPLC) (Waters; Milford, MA, USA) with a UV detector (Waters, model 2487), and a refractive index detector (Waters, model 2414) using an Aminex HPX-87(H) column (f7.8 mm 300 mm; Bio-Rad), and injecting 10 mL of sample. The eluent of 0.01 N H2SO4 was used, and analysis was performed at a flow rate of 0.7 mL/min at 508C. The retention times of metabolites were 11.6 min for glycerol, 13.1 min for acetate, 15.4 min for 1,3-PD, and 19.1 min for ethanol for the refractive index (RI), and were 10.7 min for lactate and 11.8 min for formate for UV. Calculations of metabolite concentrations were estimated from standard curves.
Results and discussion Isolation of glycerol-assimilating bacteria and selection of high ethanol producers We collected 154 soil samples from Tsukuba, Japan, and obtained 183 glycerol-assimilating strains by enrichment culture using the screening medium. To easily detect glycerol assimilation, a Durham fermentation tube was inserted into a culture vessel, and either carbon dioxide or hydrogen associated with glycerol metabolism was detected. To investigate the ethanol productivities of these isolates from glycerol, ethanol productivities were investigated using 2% glycerol basal medium (pH 7.0); the optimal condition of other known ethanol producers. As a result, a total of 115 ethanol producers were obtained with 9 high ethanol producers further isolated. Ethanol productivity also was examined under alkaline conditions (pH 9.0) because BDF-derived crude glycerol exhibits alkaline pH. Finally, one strain showed the highest productivity among all the strains obtained was selected and named TB-83. Ethanol production (120 mM; 5.5 g/L) of strain TB-83 was approximately equal under both pH conditions. In addition, consumption of glycerol increased 1.4-times at pH 9.0 (250 mM) compared to that observed at pH 7.0 (180 mM). This result indicates that glycerol was used for bacterial cell composition in the case of pH 9.0 because bacterial growth is higher than that of pH 7.0. The by-products of glycerol fermentation are 1,3-PD, lactate, acetate, and formate [7,13,14,25,26]. Strain TB-83 produced 1,3PD as the main by-product, and showed maximum ethanol production among the obtained strains under alkaline conditions. In addition, TB-83 hardly produced the other by-products, lactate and acetate, at alkaline conditions. On the basis of these results, the bacterial characteristics and ethanol productivity of the highest ethanol-producing strain, TB-83, were further examined.
Identification of strain TB-83, a glycerol-assimilating bacterium Analysis of 16S rRNA gene sequence was performed to examine the phylogeny of strain TB-83. The analysis showed that TB-83 was in the clade comprising the family Enterobacteriaceae, and revealed that the most closely related bacteria belong to the genus Klebsiella (Fig. 1a). The BLAST search of strain TB-83 revealed 99.6% similarity to the bacteria species K. variicola. 248
www.elsevier.com/locate/nbt
New Biotechnology Volume 31, Number 3 May 2014
Physiological characteristics of strainTB-83 are shown in Table 1 with K. variicola strain F2R9T (as strain ATCC BAA-830 [T = type strain]), which is a novel species related to K. pneumoniae that was first isolated from banana root in Mexico in 2004 [27]. Strain TB83, a gram-negative, non-flagella rod, oxidase negative, and catalase-positive bacterium, formed cream-colored mucoid colonies on glycerol basal agar medium (Fig. 1b). In addition, strain TB-83 showed good growth with glycerol and various sugars, and produced acids associated with their metabolism. Other phenotypic characteristics were nearly identical to the Klebsiella species [28]. The singular case was that strain F2R9T showed growth with citrate, whereas strain TB-83 did not. Strain TB-83 grew at a wide range of growth temperature, and growth pH range was comparatively high in comparison to that of strain F2R9T. It is noteworthy that the optimum pH was higher than that of other Klebsiella species [27]. Strain TB-83D exhibited the attachment of growing cells to the inner wall of the culture vessel in the glycerol basal liquid medium, and caused the agglutination with the course of cultivation (Fig. 1c). Therefore, alkaline tolerance might be attributed to cell density increased by aggregation; however, to date, Klebsiella species that grew at alkaline condition have not been reported, and the mechanism of alkaline tolerance has not been well-clarified. On the basis of the phylogenic analysis, strain TB-83 was tentatively estimated to be K. variicola, but is considered to be a new species of genus Klebsiella, by physiological characteristics.
Effect of culture pH and temperature on bacterial growth We examined the optimum pH and temperature using 2% glycerol basal medium for ethanol production. Examination of various culture temperatures revealed that temperature had little effect on product concentrations and bacterial growth (data not shown). The initial pH is very important in ethanol production from BDF waste. Bacterial growth was investigated at initial pH of 7.0, 8.0, and 9.0, and the bacteria showed hardly any difference in growth among these three pH conditions. However, the final pH decreased to around pH 6.0 at the late growth phase under all initial pH conditions, suggesting that this was caused by formate, a byproduct of ethanol production (Fig. 2a). Effects of various pH conditions were further examined at pH 5.0, 6.0, 7.0, 8,0, 9.0, and 10.0. After cultivation for 3 days, the ethanol concentration was approximately 100 mM (4.6 g/L) at pH 8.0. The concentration of formate, which was produced concomitantly with ethanol production, was 18.1 mM, 32.7 mM, 38.9 mM, and 46.0 mM at pH 7.0, 8.0, 9.0, and 10.0, respectively (Fig. 2b). The other by-products were hardly produced at any of the pH conditions evaluated. However, high and low pH conditions caused an increase in 1,3-PD production and produced 12.0 mM and 13.8 mM 1,3-PD at pH 6.0 and pH 10.0, respectively. These results revealed that the best culture pH for ethanol production was pH 8.0–9.0. For other glycerol fermentable bacteria, the initial pH for optimum ethanol production is approximately pH 7.0 [12,18]. Recently, Nwachukwu et al. reported that the best pH for ethanol production using E. aerogenes is 6.3–6.8 [29]. These results indicate that strain TB-83 has a significant advantage in the production of ethanol from alkaline BDF waste.
RESEARCH PAPER
Research Paper
New Biotechnology Volume 31, Number 3 May 2014
FIGURE 1
(a) Neighbor-joining phylogenetic tree of 16S rRNA gene sequences, showing relationship between TB-83 and the type strains that belong to the family Enterobacteriaceae. Escherichia coli served as an out-group. The numbers on the right are accession numbers in the database. The scale bar indicates 0.01 substitutions per 100 base positions. The numbers at the tree nodes are bootstrap values from 100 trials. (b) Mucoid colonies that grew on the glycerol basal agar medium after 3 days cultivation. (c) Cell aggregation that formed in glycerol basal liquid medium after 7 days cultivation.
Optimization of culture conditions for batch culture To examine the effect of nutrient sources, we added yeast extract (YE) as organic nitrogen at various concentrations using 2% glycerol basal medium at 258C for 3 days. Table 2 shows the product
concentrations and final pH at each YE concentration. The YE concentration significantly affected the production of ethanol as well as that of other metabolites. Ethanol and formate concentrations reached a maximum with the addition of 0.5 g/L YE, and
FIGURE 2
pH changes and the effect of initial pH on strain TB-83 in glycerol basal medium. (a) The relationship between pH changes and bacterial growth during ethanol fermentation. Symbols represent each pH value; circle, pH 7.0; triangle, pH 8.0; square, pH 9.0. A dotted line represents the pH changes, and a solid line represents the bacterial growth (OD580). Bacterial growth corresponds to the first longitudinal axis, and pH values correspond to the second longitudinal axis. (b) Metabolite concentrations and bacterial growth of each initial pH. Black bar, ethanol; white bar, formate; left hatched bar, 1,3-PD; right hatched bar, acetate; gray bar, lactate. Bacterial growth is represented by open circles and corresponds to the second longitudinal axis.
www.elsevier.com/locate/nbt
249
RESEARCH PAPER
New Biotechnology Volume 31, Number 3 May 2014
TABLE 1
results suggest that for efficient ethanol production, it is necessary to control pH in order to suppress 1,3-PD and formate production.
Physiological characteristics of K. variicola strains. Characteristic
Klebsiella variicola strains
Research Paper
TB-83
F2R9T (ATCC BAA-830)
Gram stain
Negative
Negative
Cell morphology
Rod shape
Rod shape
Cell dimensions (mm)
0.8 0.9–1.0
N.A.
Colony color
Cream
Yellow
Growth of Nutrient broth (NB) MacConkey Koser Christensen
+ + +
N.A. + + N.A.
Growth of anaerobic condition
+
N.A.
Flagella
Spore formation
Motility
Growth temperature range (8C) Optimum
20–45 30
N.A. 37
Growth pH range Optimum
5.0–9.0 9.0
5.6–7.0 N.A.
Utilization of Nitrate Ammonium salt Citrate Glycerol
+ + +
+ N.A. + N.A.
+, positive; , negative; N.A., data not available.
increased by approximately 1.7 and 3.7 times, respectively compared to the non-addition of YE. Further addition of YE accelerated glycerol consumption while the ethanol concentration decreased. In contrast, production of 1,3-PD, a major by-product, increased by approximately 4.0 times with the addition of YE compared to the non-addition of YE. 1,3-PD is produced from 3-hydroxypropionaldehyde (3-HPA) by coenzyme B12-dependent dehydratase, and then 3-HPA is reduced to 1,3-PD by NADH-dependent oxidoreductase [25,30]. These results imply that with the addition of YE, the increased 1,3-PD production was induced by co-enzyme B12. In particular, increased YE concentration caused a decrease in culture pH (Table 2). 1,3-PD production (approximately 5 g/L) exceeded ethanol production (approximately 3.8 g/L) with the addition of no less than 2.5 g/L YE. In addition, 1,3-PD and formate productions were inhibited at pH 6.4. Therefore, these
Ethanol production in batch cultures using two different types of BDF waste To examine ethanol productivity from BDF waste, we used two types of BDF waste (Supplementary Fig. S1a): white BDF (wBDF), which is derived from new frying oil and contains white-colored ash and very little impurities, and brown BDF waste (bBDF), which is derived from various waste-cooking oils and contains a lot of ash and impurities such as fats, oils, and triglycerides. The glycerol concentrations of wBDF and bBDF were determined as approximately 4235 mM (390 g/L) and 5581 mM (520 g/L), respectively by HPLC analysis. Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.nbt.2014.03.005. To investigate whether ethanol is produced without the inhibition by impurities, we examined media consisting of diluted BDF waste. No growth or ethanol production was observed (data not shown); therefore, we examined growth and ethanol production using 2% glycerol basal media, pH 9.0, which was replaced with BDF waste. When wBDF waste was used, the ethanol concentration reached 102 mM (4.7 g/L) and glycerol consumption was 178 mM (16.4 g/L) after 7 days cultivation (Table 3), demonstrating that BDF waste was necessary as a phosphate and nitrogen source for ethanol production. In contrast, although glycerol consumption decreased compared to wBDF waste (159 mM), ethanol productivity increased 5.4 g/L with bBDF, due to the decrease of by-products production. The molar yield from bBDF significantly increased compared to that of wBDF. Ethanol productivity was further examined using 500-mL culture vessels (Table 4). Scale-up culture without sequential addition of BDF waste caused an increase in ethanol production, and ethanol concentrations reached 188 mM (8.7 g/L) and 213 mM (9.8 g/L) after 6 and 9 days cultivation, respectively. Finally, the molar yield was 0.86 mol/mol glycerol. Nwachukwu et al. found that E. aerogenes ATCC 13048 produced 12.8 g/L ethanol from 17.8 g/L recovered glycerol acidified by H3PO4, and reported that the ethanol conversion rate was more than 70% [15]. To investigate whether the increase of ethanol production was caused by other carbon sources and fatty acids that are contained in BDF waste, we first examined total organic carbon (TOC) in the two types of BDF waste and pure glycerol. Measurement of TOC revealed that wBDF and bBDF contained 21 and 23 g/L-glycerol
TABLE 2
Effect of the addition of yeast extract (YE). YE concentration (g/L)
Products (mM)
Final pH
Consumed glycerol
Ethanol
1,3-PD
Lactate
Formate
0
102.36
58.58
15.78
0.06
13.20
6.4
0.5
134.63
97.32
19.35
1.35
49.02
5.7
1.0
166.11
93.31
39.93
1.05
36.60
5.7
2.5
216.23
82.66
62.77
6.49
24.34
5.6
5.0
269.42
78.61
61.62
3.90
6.34
5.6
Values indicate the average from two independent experiments.
250
www.elsevier.com/locate/nbt
New Biotechnology Volume 31, Number 3 May 2014
RESEARCH PAPER
TABLE 3
Metabolite productivity using wBDF and bBDF wastes after 7 days cultivation. Products (mM)
Yield (mol/mol-glycerol)
Ethanol
1,3-PD
Lactate
Formate
Acetate
Consumed glycerol
Ethanol
1,3-PD
Lactate
Formate
Acetate
wBDF
102.19
17.02
6.25
33.07
8.87
177.83
0.57
0.1
0.04
0.19
0.05
bBDF
116.11
14.69
ND
NDa
ND
159.02
0.73
0.09
ND
ND
ND
Research Paper
ND, data not detected. Values indicate the average from two independent experiments. a Formate peak in HPLC analysis was not detected.
TABLE 4
Metabolite productivity by 500 mL-scale cultivation using 2% glycerol basal medium containing bBDF. Cultivation time (days)
Products (mM)
Yield (mol/mol-glycerol)
Ethanol
1,3-PD
Lactate
Formate
Acetate
Consumed glycerol
Ethanol
1,3-PD
Lactate
Formate
Acetate
3
136.43
12.57
10.46
58.27
0.71
146.94
0.93
0.09
0.07
0.39
0.01
6
188.87
16.56
19.74
38.28
2.40
226.24
0.84
0.07
0.09
0.17
0.01
9
212.64
18.07
23.21
30.06
3.74
249.09
0.86
0.07
0.09
0.12
0.02
basal media, respectively and was higher than that of pure-glycerol (8.5 g/L-glycerol basal medium). On the other hand, GC/MS analysis showed that BDF waste contained four major fatty acids, oleic acid, linoleic acid, palmitic acid and stearic acid, although the proportion of fatty acids were almost no difference between wBDF and bBDF (Supplementary Fig. S1d). Therefore, this suggested that ethanol production of strain TB-83 was enhanced by some other component in bBDF. Table 4 shows the gradual reduction of formate concentration. Formate-hydrogen lyase (FHL) activity, which converts formate to carbon dioxide and hydrogen, is reduced under alkaline conditions. Murarka et al. showed that although the E. coli strain MG1655 produced 56 mM formate at pH 7.5, it was undetectable at pH 6.3 [31]. The reduction of formate concentrations after 6 days or 9 days cultivation suggests that formate concentration was decreased by FHL activity when shifted to neutral pH with the
accumulated formate. These findings are supported by the data shown in Fig. 2b.
Effect of sodium and potassium salts The salt concentration in BDF waste is 2.5–20 g/L [18]. Ito et al. reported that ethanol production from BDF waste significantly decreased in the presence of 3.0% NaCl [13]. They also showed that cell growth and maximum ethanol production in E. aerogenes ATCC 29007 was low at 20 g/L NaCl and KCl [18]. Ethanol production of strain TB-83 may be similarly inhibited by salts. To determine whether bacterial growth and ethanol production might be affected by sodium salts or other factors, various concentrations of NaCl and KCl were examined. A culture medium of 1% glycerol basal medium containing NaCl and KCl, pH 9.0, were used. When NaCl was used, bacterial growth was not affected until 342 mM, while ethanol production decreased significantly at 856 mM (Table 5).
TABLE 5
Effect of NaCl and KCl concentrations. Salts (g/L)
Products (mM)
OD580
Consumed glycerol
Ethanol
NaCl 0 5 10 20 30 50
103.89 106.36 105.90 98.44 69.60 3.58
98.86 102.52 106.38 97.70 86.54 7.56
KClb 0 5 10 20 30 50
109.58 84.88 97.14 81.21 66.60 35.35
97.01 86.38 101.39 95.96 86.35 52.31
1,3-PD
Lactate
Formate
7.11 5.42 4.98 4.10 3.64 0.48
3.97 3.80 4.09 4.16 5.39 0.00
37.05 39.20 39.24 37.14 34.92 9.96
1.72 1.92 1.97 1.68 1.28 0.02
11.24 7.32 9.10 7.60 6.61 4.08
8.74 4.07 5.36 5.76 6.36 6.22
30.96 33.05 33.30 32.69 31.52 18.83
1.90 2.12 1.96 1.57 1.34 0.51
a
Values indicate the average from two independent experiments. a NaCl concentrations of 0.5, 1.0, 2.0, 3.0, and 5.0% are equivalent to 86, 171, 342, 514, and 856 mM, respectively.b KCl concentrations of 0.5, 1.0, 2.0, 3.0, and 5.0% are equivalent to 67, 134, 268, 402, and 671 mM, respectively. www.elsevier.com/locate/nbt
251
RESEARCH PAPER
New Biotechnology Volume 31, Number 3 May 2014
2.5
100
2
80
1.5 60 1
OD580
Concentration
(mM)
120
40 0.5
20
Research Paper
0
0 0
1
2 3 4 5 6 7 8 9 10 Ethanol concentration (v/v%)
FIGURE 3
Bacterial growth (closed circle), consumed glycerol (open circle, dotted line), and ethanol concentration (open circle) after 24 hours cultivation of strain TB83. Bacterial growth corresponds to the second longitudinal axis. Ethanol concentration was calculated from the following equation: Actual ethanol concentration (%) = ethanol amount after 24 hours cultivation (%) initial ethanol amount (%).
medium (pH 9.0) containing 1.0, 2.0, 5.0, and 10.0% (v/v) ethanol. As shown in Fig. 3, ethanol productivity decreased by 40% with the addition of 2.0% (v/v) ethanol. With the addition of 5.0% (v/v) ethanol, no bacterial growth was observed and ethanol was not produced. This result shows that ethanol production of strain TB83 was inhibited by ethanol itself. Ethanol productivities by the mutants of other glycerol-assimilating ethanol producers exceed 20 g/L [19,20]. However, the ethanol-tolerance of these strains has not been clarified. In a single case, E. coli strains LY01, LY02, and LY03 mutated toward increased ethanol-tolerance, and produced more ethanol than did the wild strain [35]. This increased ethanol production may be due to improvement of ethanol-tolerance by the mutation. Molecular breeding by selecting for natural mutations is in progress to obtain other high ethanol producers and high ethanol-tolerant strains.
Conclusions
On the one hand, the addition of KCl showed no difference in comparison to the effect of NaCl (Table 5). Gonzalez et al. reported that bacterial growth and glycerol fermentation were inhibited in the presence of potassium ions [32]. However, bacterial growth was inhibited by the addition of 671 mM KCl, and ethanol production of strain TB-83 was not affected at less than 402 mM KCl. In both cases, although ethanol productivity slightly decreased from 20 g/ L of NaCl and KCl onwards, the molar yield finally increased. Since BDF waste that diluted 20 times with distilled water was used, salt concentration containing the media was be estimated to be at less than 10 g/L. These results indicate that ethanol production is not barely affected by salts such as NaCl and KCl at less than 20 g/L.
In this study, we obtained a novel high potential strain TB-83 that showed maximum ethanol productivity at alkaline pH. In addition, waste cooking oil-derived BDF, bBDF, facilitated higher ethanol production compared to wBDF, which is a new frying oil-derived BDF. Investigation of culture conditions demonstrated that the addition of yeast extract was highly effective in producing ethanol. However, the use of yeast extract in ethanol production from BDF waste is difficult to economically in terms of production costs, therefore, we need to examine the investigation of media component considering the production costs, in addition to the further improvement in ethanol production. To overcome this problem, we have been currently studying further investigation of a media component for the reduction of production costs. In addition, a further improved ethanol-tolerant strain obtained by mutation and scaled-up culture by pH control will be required, to further improve ethanol production.
Ethanol-tolerance
Acknowledgments
In ethanol production, it has been reported that low ethanol tolerance of bacteria is one of the major limiting factors for ethanol production [33,34]. Therefore, it is possible that ethanol production of strain TB-83 is affected by the ethanol concentration in the culture. To evaluate the ethanol tolerance of strain TB-83 at various ethanol concentrations, we used 1% glycerol basal
We thank Dr Masayuki Onodera (Niigata Institute of Technology) and Nagasaki Prefectural Institute for Environmental Research and Public Health for providing BDF waste. This work was supported by the Environment Research and Technology Development Fund (ERTDF) (3K123007) from the Ministry of the Environment, Government of Japan.
References [1] Ma F, Hanna MA. Biodiesel production: a review. Bioresour Technol 1999;70:1– 15. [2] He H, Wang T, Zhu S. Continuous production of biodiesel fuel from vegetable oil using supercritical methanol process. Fuel 2007;86:442–7. [3] Sakai S, Yagishita T. Microbial production of hydrogen and ethanol from glycerolcontaining wastes discharged from a biodiesel fuel production plant in a bioelectrochemical reactor with thionine. Biotechnol Bioeng 2007;98:340–8. [4] Ranganathan SV, Narasimhan SL, Muthukumar K. An overview of enzymatic production of biodiesel. Bioresour Technol 2008;99:3975–81. [5] Fukuda H, Kondo A, Noda H. Biodiesel fuel production by transesterification of oils. J Biosci Bioeng 2001;92:405–16. [6] Johnson DT, Taconi KA. The glycerin glut: options for the value-added conversion of crude glycerol resulting from biodiesel production. Environ Prog 2007;26:338–48. [7] da Silva GP, Mack M, Contiero J. Glycerol: a promising and abundant carbon source for industrial microbiology. Biotechnol Adv 2009;27:30–9. [8] Cheng KK, Liu HJ, Liu DH. Multiple growth inhibition of Klebsiella pneumoniae in 1,3-propanediol fermentation. Biotechnol Lett 2005;27:19–22.
252
www.elsevier.com/locate/nbt
[9] Mu Y, Teng H, Zhang DJ, Wang W, Xiu ZL. Microbial production of 1,3propanediol by Klebsiella pneumoniae using crude glycerol from biodiesel preparations. Biotechnol Lett 2006;28:1124–35. [10] Biebl H. Glycerol fermentation of 1,3-propanediol by Clostridium butyricum measurement of product inhibition by use of pH-auxostat. Appl Microbiol Biotechnol 1991;35:701–5. [11] Gonza´lez-Pajuelo M, Meynial-Salles I, Mendes F, Soucaille P, Vasconcelos I. Microbial conversion of glycerol to 1,3-propanediol: physiological comparison of a natural producer, Clostridium butyricum VPI 3266, and an engineered strain, Clostridium acetobutylicum DG1(pSPD5). Appl Environ Microbiol 2006;72:96–101. [12] Ito T, Nakashimada Y, Kakizono T, Nishio N. High-yield production of hydrogen by Enterobacter aerogenes mutants with decreased alpha-acetolactate synthase activity. J Biosci Bioeng 2004;97:27–232. [13] Ito T, Nakashimada Y, Senba K, Matsui T, Nishio N. Hydrogen and ethanol production from glycerol-containing wastes discharged after biodiesel manufacturing process. J Biosci Bioeng 2005;100:260–5. ¨ A, Santala V, Karp M. Hydrogen production from glycerol using halo[14] Kivisto philic fermentative bacteria. Bioresour Technol 2010;101:8671–7.
[15] Nwachukwu R, Shahbazi A, Wang L, Ibrahim S, Worku M, Schimmel K. Bioconversion of glycerol to ethanol by a mutant Enterobacter aerogenes. AMB Express 2012;2:20. [16] Jarvis GN, Moore ER, Thiele JH. Formate and ethanol are the major products of glycerol fermentation produced by a Klebsiella planticola strain isolated from red deer. J Appl Microbiol 1997;83:166–74. [17] Taconi KA, Venkataramanan KP, Johnson DT. Growth and solvent production by Clostridium pasteurianum ATCC 6013 utilizing biodiesel-derived crude glycerol as the sole carbon source. Environ Prog Sust Energy 2009;28:100–10. [18] Lee SJ, Kim SB, Kang SW, Han SO, Park C, Kim SW. Effect of crude glycerolderived inhibitors on ethanol production by Enterobacter aerogenes. Bioprocess Biosyst Eng 2012;35:85–92. [19] Oh BR, Seo JW, Heo SY, Hong WK, Luo LH, Joe MH, et al. Efficient production of ethanol from crude glycerol by a Klebsiella pneumoniae mutant strain. Bioresour Technol 2011;102:3918–22. [20] Saisaard K, Angelidaki I, Prasertsan P. Micro-aerobic, anaerobic and two-stage condition for ethanol production by Enterobacter aerogenes from biodieselderived crude glycerol. World Acad Sci Eng Technol 2011;53:795–8. [21] Choi WJ, Hartono MR, Chan WH, Yeo SS. Ethanol production from biodieselderived crude glycerol by newly isolated Kluyvera cryocrescens. Appl Microbiol Biotechnol 2011;89:1255–64. [22] Ban K, Kaieda M, Matsumoto T, Kondo A, Fukuda H. Whole cell biocatalyst for biodiesel fuel production utilizing Rhizopus oryzae cells immobilized within biomass support particles. Biochem Eng J 2001;8:39–43. [23] Sambrook J, Russell DW. Molecular cloning: a laboratory manual, 3rd ed., Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 2001. [24] Lane DJ. 16S/23S rRNA sequencing. In: Stackebrandt E, Goodfellow M, editors. Nucleic acid techniques in bacterial systematics. New York: Wiley; 1991. p. 115–47. [25] Tong I, Cameron D. Enhancement of 1,3-propanediol production by cofermentation in Escherichia coli expressing genes from Klebsiella pneumoniae dha regulon genes. Appl Biochem Biotechnol 1992;34/35:149–59.
RESEARCH PAPER
[26] Durnin G, Clomburg J, Yeates Z, Alvarez PJJ, Zygourakis K, Campbell P, et al. Understanding and harnessing the microaerobic metabolism of glycerol in Escherichia coli. Biotechnol Bioeng 2009;103:148–61. [27] Rosenblueth M, Martinez L, Silva J, Martinez-Romero E. Klebsiella variicola, a novel species with clinical and plant-associated isolates. Syst Appl Microbiol 2004;27:27–35. [28] Brisse S, Grimont F, Grimont PAD. The genus Klebsiella. Prokaryotes 2006;6:159– 96. [29] Nwachukwu RE, Shahbazi A, Wang L, Worku M, Ibrahim S, Schimmel K. Optimization of cultural conditions for conversion of glycerol to ethanol by Enterobacter aerogenes S012. AMB Express 2013;3:12. [30] Ashok S, Raj SM, Rathnasingh C, Park S. Development of recombinant Klebsiella pneumoniae DdhaT strain for the co-production of 3-hydroxypropionic acid and 1,3-propanediol from glycerol. Appl Microbiol Biotechnol 2011;90: 1253–65. [31] Murarka A, Dharmadi Y, Yazdani SS, Gonzalez R. Fermentative utilization of glycerol in Escherichia coli and its implications for the production of reduced chemicals and fuels. Appl Environ Microbiol 2008;74:1124–35. [32] Gonzalez R, Murarka A, Dharmadi Y, Yazdani SS. A new model for the anaerobic fermentation of glycerol in enteric bacteria: trunk and auxiliary pathways in Escherichia coli. Metab Eng 2008;10:234–45. [33] Sudha Rani K, Seenayya G. High ethanol tolerance of new isolates of Clostridium thermocellum strains SS21 and SS22. World J Microbiol Biotechnol 1999;15:173– 8. [34] Georgieva TI, Mikkelsen MJ, Ahring BK. High ethanol tolerance of the thermophilic anaerobic ethanol producer Thermoanaerobacter BG1L1. Cent Eur J Biol 2007;2:364–77. [35] Yomano LP, York SW, Ingram LO. Isolation and characterization of ethanol tolerant mutants of Escherichia coli KO11 for fuel ethanol production. J Ind Microbiol Biotechnol 1998;20:32–138.
www.elsevier.com/locate/nbt
253
Research Paper
New Biotechnology Volume 31, Number 3 May 2014
New Biotechnology Volume 31, Number 3 May 2014
Research Paper
Ethanol production from glycerolcontaining biodiesel waste by Klebsiella variicola shows maximum productivity under alkaline conditions Toshihiro Suzuki1, Chiaki Nishikawa1, Kohei Seta1, Toshiya Shigeno2 and Toshiaki Nakajima-Kambe1 1 2
Faculty of Life and Environmental Sciences (Bioindustrial Sciences), University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan Tsukuba Environmental Microorganism Institute, 8-1 Sakuragaoka, Tsukuba, Ibaraki 300-1271, Japan
Biodiesel fuel (BDF) waste contains large amounts of crude glycerol as a by-product, and has a high alkaline pH. With regard to microbial conversion of ethanol from BDF-derived glycerol, bacteria that can produce ethanol at alkaline pH have not been reported to date. Isolation of bacteria that shows maximum productivity under alkaline conditions is essential to effective production of ethanol from BDF-derived glycerol. In this study, we isolated the Klebsiella variicola TB-83 strain, which demonstrated maximum ethanol productivity at alkaline pH. Strain TB-83 showed effective usage of crude glycerol with maximum ethanol production at pH 8.0–9.0, and the culture pH was finally neutralized by formate, a by-product. In addition, the ethanol productivity of strain TB-83 under various culture conditions was investigated. Ethanol production was more efficient with the addition of yeast extract. Strain TB-83 produced 9.8 g/L ethanol (0.86 mol/mol glycerol) from cooking oil-derived BDF waste. Ethanol production from cooking oil-derived BDF waste was higher than that of new frying oil-derived BDF and pure-glycerol. This is the first report to demonstrate that the K. variicola strain TB-83 has the ability to produce ethanol from glycerol at alkaline pH.
Introduction Biodiesel fuel (BDF) is biomass fuel that is used as an alternative fuel of diesel oil. BDF, a carbon neutral fuel, is also made from vegetable oils and animal fats [1–4]. Almost all BDF production is carried out by transesterification with methanol in the presence of an alkaline catalyst [3,5]. However, BDF waste has a high alkaline pH, and contains a large amount of crude glycerol as a by-product [6]. Since almost all BDF-derived glycerol is waste oil-derived, it has no applied use, and is almost always incinerated. Therefore, the treatment of BDF-derived crude glycerol is an environmental problem. Bacteria that can utilize glycerol as a sole carbon source are known to produce some valuable metabolites [3,7]. Recent studies have reported that the major metabolites derived from glycerol-assimilation are 1,3-propanediol (1,3-PD) [8–11], hydrogen [12–14], and
Corresponding author: Nakajima-Kambe, T. ([email protected]) www.elsevier.com/locate/nbt
246
ethanol [13,15,16]. Bio-ethanol is particularly valuable as its biomass energy as well as BDF. The production of bio-ethanol is an efficient use of waste and decreases burdens on the environment. In the fermentation of glycerol, the initial culture pH is neutral but gradually decreases through the accumulation of organic acid by-products [12–17]. The same problem of glycerol residue treatment also emerges in the case of BDF. In addition, when pure glycerol is used, many ethanol producers show high ethanol production, whereas ethanol productivity decreases using BDF waste [13,18]. Recent studies of BDF waste have revealed that the family Enterobacteriaceae and its mutant strains produce higher amounts of ethanol when using BDF than pure glycerol. Klebsiella pneumoniae GEM167, a gamma-irradiation mutant, produces 25.0 g/L of ethanol with a productivity rate of 0.93 g L1 hour1 from BDF waste [19]. Saisaard et al. reported that Enterobacter aerogenes TISTR1468 produced 24.5 g/L using waste
http://dx.doi.org/10.1016/j.nbt.2014.03.005 1871-6784/ß 2014 Elsevier B.V. All rights reserved.
New Biotechnology Volume 31, Number 3 May 2014
Materials and methods Media and reagents Screening medium containing 10.0 g KH2PO4, 1.0 g NH4Cl, 0.50 g yeast extract (Difco; Detroit, MI, USA), and 100.0 g glycerol in 1 L of distilled water (adjusted to pH 7.0 with 6 N NaOH) was used for screening bacteria. Glycerol basal medium, containing 10.0 g KH2PO4, 1.0 g NH4Cl, 0.50 g yeast extract (Difco) 1.0 g casamino acid (Difco), and 20.0 g glycerol in 1 L of distilled water, was used for isolating and culturing bacteria. When examining BDF waste, glycerol was replaced with BDF waste. If not otherwise specified, all reagents for this study were obtained from Wako pure chemical industries, Ltd (Osaka, Japan). Two types of BDF waste were provided: BDF waste containing white-colored ash and very little impurities (Niigata Institute of Technology), which is a new frying oil-derived BDF, and brown BDF waste containing a lot of ash and impurities such as fats, oils, and triglycerides, which is a waste cooking oil-derived BDF (Nagasaki Prefectural Institute for Environmental Research and Public Health). Total organic carbon (TOC) containing the two types of BDF waste were analyzed with Shimadzu TOC-V, a total organic carbon analyzer (Shimadzu, Kyoto, Japan), and glycerol concentration was measured with high-performance liquid chromatography (HPLC). To examine the fatty acids composition, BDF waste (20 mg) was methyl-esterified with 5% hydrogen chloride methanol solution (Wako) at 958C for 3 hours, and then extracted with n-hexane. The n-hexane layer was analyzed by GC/MS, TRACE GC gas chromatography and FINNIGAN POLARIS Q mass spectrometer (Thermo Scientific, Tokyo, Japan) equipped with a DB-1MS fused Silica Capillary column (30 m 0.25 mm 0.25 mm) (Agilent Technologies, Inc., Fort Collins, CO, USA), and helium was used as career gas. Analysis was performed according to the method of Ban et al. [22].
Sampling and isolation of bacteria Soil samples (500 mg) from Tsukuba, Japan were collected in test tubes (f25 mm 200 mm) and cultured at 258C in 30 mL of the screening medium inserted into a Durham fermentation tube (f9 mm 30 mm). After bubbles were observed in the Durham fermentation tube, 0.5 mL of the broth was inoculated into the same medium. This isolation procedure was repeated at least three times. A portion of the broth was streaked onto glycerol basal agar medium and cultured at 308C for 7 days. Determination of bio-
chemical characteristic was performed by TechnoSuruga Laboratory Co., Ltd. (Shizuoka, Japan).
Genetic manipulations Total DNA isolation from Klebsiella variicola TB-83 was conducted as described below. Cells were harvested from 5 mL nutrient broth (NB) (Difco) liquid medium by centrifugation and washed twice with 567 mL TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0). Cells were re-suspended in same volume of TE buffer to which 30 mL 10% sodium dodecyl sulfate and 3 mL Proteinase K solution (20 mg/mL in TE) were added followed by incubation at 378C for 1 hour. NaCl (2 mL; 5 M) was added followed by mixing for 30 min, and then 80 mL hexadecyltrimethyl ammonium bromide (CTAB)/NaCl solution was added to the cell suspension. The mixture was incubated at 658C for 10 min, and then chloroform:isoamyl alcohol (24:1) was added followed by shaking. The mixture was centrifuged at 7000 g for 5 min and then the supernatant was isopropanol-precipitated. The precipitate was dissolved in TE buffer, extracted with phenol, incubated with RNase (100 mg/mL) at 378C for 1 hour, and extracted again with phenol:chloroform:isoamyl alcohol (25:24:1). DNA manipulations of Escherichia coli were performed according to Sambrook et al. [23]. The 16S rRNA gene coding sequence was amplified from total DNA as a template using primers 27F (50 -AGAGTTTGATCCTGGCTCAG-30 ) and 1492R (50 -TGACTGACTGAGGYTACCTTGTTACGACTT-30 ) [24]. Polymerase chain reaction (PCR) amplification was performed with the MJ Mini Personal Thermal Cycler (Bio-Rad; Hercules, CA) using Ex Taq DNA polymerase (Takara; Kyoto, Japan). PCR was conducted as follows: 948C for 2 min, followed by 30 cycles of 988C for 15 s, 608C for 20 s, and 748C for 1 min. The PCR product was ligated into the pGEM-T Easy vector (Promega; Madison, WI, USA) for sequence analysis, and then transformed in E. coli DH10B as a host strain. DNA sequencing was carried out by cycle sequencing using the BigDye terminator v3.1 cycle sequencing kit (Applied Biosystems; Foster City, CA, USA) and analyzed with an ABI PRISMTM 3130NT genetic analyzer (Applied Biosystems).
Phylogenetic analysis Homology searches were conducted using the BLAST program (http://www.ncbi.nlm.nih.gov/BLAST/). The sequences were aligned using the CLUSTALW ver. 1.83 program. Phylogenetic trees were constructed using TreeViewX software by the neighborjoining method. A bootstrap analysis with 100 trial replications was performed to determine the reliability of clustering patterns.
Culture conditions for ethanol production Ethanol production was performed using glycerol basal medium under various pH, temperature, and culture conditions. One loop of strain TB-83 grown on an NB agar plate was suspended with 1 mL of glycerol basal liquid medium. The suspension was inoculated into a test tube (f25 mm 200 mm) containing 30 mL of glycerol basal medium (final OD580 = 0.01) and incubated for various periods at 258C under static culture. One milliliter of culture was sequentially collected and analyzed for OD580, culture pH, and product concentrations. To examine batch cultures in basal medium using BDF waste, glycerol was replaced with BDF waste diluted 20 times with distilled water. www.elsevier.com/locate/nbt
247
Research Paper
cooking oil-derived BDF [20]. Recently, Kluyvera cryocrescens S26, a wild strain, was shown to produce 27 g/L with 80% yield by batch culture of palm oil-derived crude glycerol [21]. However, bacteria that can produce ethanol at alkaline pH have not been reported. In addition to the identification of bacteria that can produce ethanol at alkaline pH, evaluation of ethanol productivity is essential for high-efficiency ethanol production using BDF waste from the standpoint of production costs. To overcome the problem of ethanol production at alkaline pH, we isolated glycerol-assimilating bacteria from a soil environment and investigated their ethanol productivities at alkaline pH under various culture conditions. In addition, effects of the culture components in ethanol production were examined.
RESEARCH PAPER
RESEARCH PAPER
Measurement of metabolite concentrations
Research Paper
The supernatant of 1 mL culture was filtered immediately through a 0.2-mm polyvinylidene fluoride single-step filter vial (Thomson Instrument Company; Oceanside, CA, USA). Ethanol and other metabolite concentrations were measured by Waters alliance 2695 high-performance liquid chromatography (HPLC) (Waters; Milford, MA, USA) with a UV detector (Waters, model 2487), and a refractive index detector (Waters, model 2414) using an Aminex HPX-87(H) column (f7.8 mm 300 mm; Bio-Rad), and injecting 10 mL of sample. The eluent of 0.01 N H2SO4 was used, and analysis was performed at a flow rate of 0.7 mL/min at 508C. The retention times of metabolites were 11.6 min for glycerol, 13.1 min for acetate, 15.4 min for 1,3-PD, and 19.1 min for ethanol for the refractive index (RI), and were 10.7 min for lactate and 11.8 min for formate for UV. Calculations of metabolite concentrations were estimated from standard curves.
Results and discussion Isolation of glycerol-assimilating bacteria and selection of high ethanol producers We collected 154 soil samples from Tsukuba, Japan, and obtained 183 glycerol-assimilating strains by enrichment culture using the screening medium. To easily detect glycerol assimilation, a Durham fermentation tube was inserted into a culture vessel, and either carbon dioxide or hydrogen associated with glycerol metabolism was detected. To investigate the ethanol productivities of these isolates from glycerol, ethanol productivities were investigated using 2% glycerol basal medium (pH 7.0); the optimal condition of other known ethanol producers. As a result, a total of 115 ethanol producers were obtained with 9 high ethanol producers further isolated. Ethanol productivity also was examined under alkaline conditions (pH 9.0) because BDF-derived crude glycerol exhibits alkaline pH. Finally, one strain showed the highest productivity among all the strains obtained was selected and named TB-83. Ethanol production (120 mM; 5.5 g/L) of strain TB-83 was approximately equal under both pH conditions. In addition, consumption of glycerol increased 1.4-times at pH 9.0 (250 mM) compared to that observed at pH 7.0 (180 mM). This result indicates that glycerol was used for bacterial cell composition in the case of pH 9.0 because bacterial growth is higher than that of pH 7.0. The by-products of glycerol fermentation are 1,3-PD, lactate, acetate, and formate [7,13,14,25,26]. Strain TB-83 produced 1,3PD as the main by-product, and showed maximum ethanol production among the obtained strains under alkaline conditions. In addition, TB-83 hardly produced the other by-products, lactate and acetate, at alkaline conditions. On the basis of these results, the bacterial characteristics and ethanol productivity of the highest ethanol-producing strain, TB-83, were further examined.
Identification of strain TB-83, a glycerol-assimilating bacterium Analysis of 16S rRNA gene sequence was performed to examine the phylogeny of strain TB-83. The analysis showed that TB-83 was in the clade comprising the family Enterobacteriaceae, and revealed that the most closely related bacteria belong to the genus Klebsiella (Fig. 1a). The BLAST search of strain TB-83 revealed 99.6% similarity to the bacteria species K. variicola. 248
www.elsevier.com/locate/nbt
New Biotechnology Volume 31, Number 3 May 2014
Physiological characteristics of strainTB-83 are shown in Table 1 with K. variicola strain F2R9T (as strain ATCC BAA-830 [T = type strain]), which is a novel species related to K. pneumoniae that was first isolated from banana root in Mexico in 2004 [27]. Strain TB83, a gram-negative, non-flagella rod, oxidase negative, and catalase-positive bacterium, formed cream-colored mucoid colonies on glycerol basal agar medium (Fig. 1b). In addition, strain TB-83 showed good growth with glycerol and various sugars, and produced acids associated with their metabolism. Other phenotypic characteristics were nearly identical to the Klebsiella species [28]. The singular case was that strain F2R9T showed growth with citrate, whereas strain TB-83 did not. Strain TB-83 grew at a wide range of growth temperature, and growth pH range was comparatively high in comparison to that of strain F2R9T. It is noteworthy that the optimum pH was higher than that of other Klebsiella species [27]. Strain TB-83D exhibited the attachment of growing cells to the inner wall of the culture vessel in the glycerol basal liquid medium, and caused the agglutination with the course of cultivation (Fig. 1c). Therefore, alkaline tolerance might be attributed to cell density increased by aggregation; however, to date, Klebsiella species that grew at alkaline condition have not been reported, and the mechanism of alkaline tolerance has not been well-clarified. On the basis of the phylogenic analysis, strain TB-83 was tentatively estimated to be K. variicola, but is considered to be a new species of genus Klebsiella, by physiological characteristics.
Effect of culture pH and temperature on bacterial growth We examined the optimum pH and temperature using 2% glycerol basal medium for ethanol production. Examination of various culture temperatures revealed that temperature had little effect on product concentrations and bacterial growth (data not shown). The initial pH is very important in ethanol production from BDF waste. Bacterial growth was investigated at initial pH of 7.0, 8.0, and 9.0, and the bacteria showed hardly any difference in growth among these three pH conditions. However, the final pH decreased to around pH 6.0 at the late growth phase under all initial pH conditions, suggesting that this was caused by formate, a byproduct of ethanol production (Fig. 2a). Effects of various pH conditions were further examined at pH 5.0, 6.0, 7.0, 8,0, 9.0, and 10.0. After cultivation for 3 days, the ethanol concentration was approximately 100 mM (4.6 g/L) at pH 8.0. The concentration of formate, which was produced concomitantly with ethanol production, was 18.1 mM, 32.7 mM, 38.9 mM, and 46.0 mM at pH 7.0, 8.0, 9.0, and 10.0, respectively (Fig. 2b). The other by-products were hardly produced at any of the pH conditions evaluated. However, high and low pH conditions caused an increase in 1,3-PD production and produced 12.0 mM and 13.8 mM 1,3-PD at pH 6.0 and pH 10.0, respectively. These results revealed that the best culture pH for ethanol production was pH 8.0–9.0. For other glycerol fermentable bacteria, the initial pH for optimum ethanol production is approximately pH 7.0 [12,18]. Recently, Nwachukwu et al. reported that the best pH for ethanol production using E. aerogenes is 6.3–6.8 [29]. These results indicate that strain TB-83 has a significant advantage in the production of ethanol from alkaline BDF waste.
RESEARCH PAPER
Research Paper
New Biotechnology Volume 31, Number 3 May 2014
FIGURE 1
(a) Neighbor-joining phylogenetic tree of 16S rRNA gene sequences, showing relationship between TB-83 and the type strains that belong to the family Enterobacteriaceae. Escherichia coli served as an out-group. The numbers on the right are accession numbers in the database. The scale bar indicates 0.01 substitutions per 100 base positions. The numbers at the tree nodes are bootstrap values from 100 trials. (b) Mucoid colonies that grew on the glycerol basal agar medium after 3 days cultivation. (c) Cell aggregation that formed in glycerol basal liquid medium after 7 days cultivation.
Optimization of culture conditions for batch culture To examine the effect of nutrient sources, we added yeast extract (YE) as organic nitrogen at various concentrations using 2% glycerol basal medium at 258C for 3 days. Table 2 shows the product
concentrations and final pH at each YE concentration. The YE concentration significantly affected the production of ethanol as well as that of other metabolites. Ethanol and formate concentrations reached a maximum with the addition of 0.5 g/L YE, and
FIGURE 2
pH changes and the effect of initial pH on strain TB-83 in glycerol basal medium. (a) The relationship between pH changes and bacterial growth during ethanol fermentation. Symbols represent each pH value; circle, pH 7.0; triangle, pH 8.0; square, pH 9.0. A dotted line represents the pH changes, and a solid line represents the bacterial growth (OD580). Bacterial growth corresponds to the first longitudinal axis, and pH values correspond to the second longitudinal axis. (b) Metabolite concentrations and bacterial growth of each initial pH. Black bar, ethanol; white bar, formate; left hatched bar, 1,3-PD; right hatched bar, acetate; gray bar, lactate. Bacterial growth is represented by open circles and corresponds to the second longitudinal axis.
www.elsevier.com/locate/nbt
249
RESEARCH PAPER
New Biotechnology Volume 31, Number 3 May 2014
TABLE 1
results suggest that for efficient ethanol production, it is necessary to control pH in order to suppress 1,3-PD and formate production.
Physiological characteristics of K. variicola strains. Characteristic
Klebsiella variicola strains
Research Paper
TB-83
F2R9T (ATCC BAA-830)
Gram stain
Negative
Negative
Cell morphology
Rod shape
Rod shape
Cell dimensions (mm)
0.8 0.9–1.0
N.A.
Colony color
Cream
Yellow
Growth of Nutrient broth (NB) MacConkey Koser Christensen
+ + +
N.A. + + N.A.
Growth of anaerobic condition
+
N.A.
Flagella
Spore formation
Motility
Growth temperature range (8C) Optimum
20–45 30
N.A. 37
Growth pH range Optimum
5.0–9.0 9.0
5.6–7.0 N.A.
Utilization of Nitrate Ammonium salt Citrate Glycerol
+ + +
+ N.A. + N.A.
+, positive; , negative; N.A., data not available.
increased by approximately 1.7 and 3.7 times, respectively compared to the non-addition of YE. Further addition of YE accelerated glycerol consumption while the ethanol concentration decreased. In contrast, production of 1,3-PD, a major by-product, increased by approximately 4.0 times with the addition of YE compared to the non-addition of YE. 1,3-PD is produced from 3-hydroxypropionaldehyde (3-HPA) by coenzyme B12-dependent dehydratase, and then 3-HPA is reduced to 1,3-PD by NADH-dependent oxidoreductase [25,30]. These results imply that with the addition of YE, the increased 1,3-PD production was induced by co-enzyme B12. In particular, increased YE concentration caused a decrease in culture pH (Table 2). 1,3-PD production (approximately 5 g/L) exceeded ethanol production (approximately 3.8 g/L) with the addition of no less than 2.5 g/L YE. In addition, 1,3-PD and formate productions were inhibited at pH 6.4. Therefore, these
Ethanol production in batch cultures using two different types of BDF waste To examine ethanol productivity from BDF waste, we used two types of BDF waste (Supplementary Fig. S1a): white BDF (wBDF), which is derived from new frying oil and contains white-colored ash and very little impurities, and brown BDF waste (bBDF), which is derived from various waste-cooking oils and contains a lot of ash and impurities such as fats, oils, and triglycerides. The glycerol concentrations of wBDF and bBDF were determined as approximately 4235 mM (390 g/L) and 5581 mM (520 g/L), respectively by HPLC analysis. Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.nbt.2014.03.005. To investigate whether ethanol is produced without the inhibition by impurities, we examined media consisting of diluted BDF waste. No growth or ethanol production was observed (data not shown); therefore, we examined growth and ethanol production using 2% glycerol basal media, pH 9.0, which was replaced with BDF waste. When wBDF waste was used, the ethanol concentration reached 102 mM (4.7 g/L) and glycerol consumption was 178 mM (16.4 g/L) after 7 days cultivation (Table 3), demonstrating that BDF waste was necessary as a phosphate and nitrogen source for ethanol production. In contrast, although glycerol consumption decreased compared to wBDF waste (159 mM), ethanol productivity increased 5.4 g/L with bBDF, due to the decrease of by-products production. The molar yield from bBDF significantly increased compared to that of wBDF. Ethanol productivity was further examined using 500-mL culture vessels (Table 4). Scale-up culture without sequential addition of BDF waste caused an increase in ethanol production, and ethanol concentrations reached 188 mM (8.7 g/L) and 213 mM (9.8 g/L) after 6 and 9 days cultivation, respectively. Finally, the molar yield was 0.86 mol/mol glycerol. Nwachukwu et al. found that E. aerogenes ATCC 13048 produced 12.8 g/L ethanol from 17.8 g/L recovered glycerol acidified by H3PO4, and reported that the ethanol conversion rate was more than 70% [15]. To investigate whether the increase of ethanol production was caused by other carbon sources and fatty acids that are contained in BDF waste, we first examined total organic carbon (TOC) in the two types of BDF waste and pure glycerol. Measurement of TOC revealed that wBDF and bBDF contained 21 and 23 g/L-glycerol
TABLE 2
Effect of the addition of yeast extract (YE). YE concentration (g/L)
Products (mM)
Final pH
Consumed glycerol
Ethanol
1,3-PD
Lactate
Formate
0
102.36
58.58
15.78
0.06
13.20
6.4
0.5
134.63
97.32
19.35
1.35
49.02
5.7
1.0
166.11
93.31
39.93
1.05
36.60
5.7
2.5
216.23
82.66
62.77
6.49
24.34
5.6
5.0
269.42
78.61
61.62
3.90
6.34
5.6
Values indicate the average from two independent experiments.
250
www.elsevier.com/locate/nbt
New Biotechnology Volume 31, Number 3 May 2014
RESEARCH PAPER
TABLE 3
Metabolite productivity using wBDF and bBDF wastes after 7 days cultivation. Products (mM)
Yield (mol/mol-glycerol)
Ethanol
1,3-PD
Lactate
Formate
Acetate
Consumed glycerol
Ethanol
1,3-PD
Lactate
Formate
Acetate
wBDF
102.19
17.02
6.25
33.07
8.87
177.83
0.57
0.1
0.04
0.19
0.05
bBDF
116.11
14.69
ND
NDa
ND
159.02
0.73
0.09
ND
ND
ND
Research Paper
ND, data not detected. Values indicate the average from two independent experiments. a Formate peak in HPLC analysis was not detected.
TABLE 4
Metabolite productivity by 500 mL-scale cultivation using 2% glycerol basal medium containing bBDF. Cultivation time (days)
Products (mM)
Yield (mol/mol-glycerol)
Ethanol
1,3-PD
Lactate
Formate
Acetate
Consumed glycerol
Ethanol
1,3-PD
Lactate
Formate
Acetate
3
136.43
12.57
10.46
58.27
0.71
146.94
0.93
0.09
0.07
0.39
0.01
6
188.87
16.56
19.74
38.28
2.40
226.24
0.84
0.07
0.09
0.17
0.01
9
212.64
18.07
23.21
30.06
3.74
249.09
0.86
0.07
0.09
0.12
0.02
basal media, respectively and was higher than that of pure-glycerol (8.5 g/L-glycerol basal medium). On the other hand, GC/MS analysis showed that BDF waste contained four major fatty acids, oleic acid, linoleic acid, palmitic acid and stearic acid, although the proportion of fatty acids were almost no difference between wBDF and bBDF (Supplementary Fig. S1d). Therefore, this suggested that ethanol production of strain TB-83 was enhanced by some other component in bBDF. Table 4 shows the gradual reduction of formate concentration. Formate-hydrogen lyase (FHL) activity, which converts formate to carbon dioxide and hydrogen, is reduced under alkaline conditions. Murarka et al. showed that although the E. coli strain MG1655 produced 56 mM formate at pH 7.5, it was undetectable at pH 6.3 [31]. The reduction of formate concentrations after 6 days or 9 days cultivation suggests that formate concentration was decreased by FHL activity when shifted to neutral pH with the
accumulated formate. These findings are supported by the data shown in Fig. 2b.
Effect of sodium and potassium salts The salt concentration in BDF waste is 2.5–20 g/L [18]. Ito et al. reported that ethanol production from BDF waste significantly decreased in the presence of 3.0% NaCl [13]. They also showed that cell growth and maximum ethanol production in E. aerogenes ATCC 29007 was low at 20 g/L NaCl and KCl [18]. Ethanol production of strain TB-83 may be similarly inhibited by salts. To determine whether bacterial growth and ethanol production might be affected by sodium salts or other factors, various concentrations of NaCl and KCl were examined. A culture medium of 1% glycerol basal medium containing NaCl and KCl, pH 9.0, were used. When NaCl was used, bacterial growth was not affected until 342 mM, while ethanol production decreased significantly at 856 mM (Table 5).
TABLE 5
Effect of NaCl and KCl concentrations. Salts (g/L)
Products (mM)
OD580
Consumed glycerol
Ethanol
NaCl 0 5 10 20 30 50
103.89 106.36 105.90 98.44 69.60 3.58
98.86 102.52 106.38 97.70 86.54 7.56
KClb 0 5 10 20 30 50
109.58 84.88 97.14 81.21 66.60 35.35
97.01 86.38 101.39 95.96 86.35 52.31
1,3-PD
Lactate
Formate
7.11 5.42 4.98 4.10 3.64 0.48
3.97 3.80 4.09 4.16 5.39 0.00
37.05 39.20 39.24 37.14 34.92 9.96
1.72 1.92 1.97 1.68 1.28 0.02
11.24 7.32 9.10 7.60 6.61 4.08
8.74 4.07 5.36 5.76 6.36 6.22
30.96 33.05 33.30 32.69 31.52 18.83
1.90 2.12 1.96 1.57 1.34 0.51
a
Values indicate the average from two independent experiments. a NaCl concentrations of 0.5, 1.0, 2.0, 3.0, and 5.0% are equivalent to 86, 171, 342, 514, and 856 mM, respectively.b KCl concentrations of 0.5, 1.0, 2.0, 3.0, and 5.0% are equivalent to 67, 134, 268, 402, and 671 mM, respectively. www.elsevier.com/locate/nbt
251
RESEARCH PAPER
New Biotechnology Volume 31, Number 3 May 2014
2.5
100
2
80
1.5 60 1
OD580
Concentration
(mM)
120
40 0.5
20
Research Paper
0
0 0
1
2 3 4 5 6 7 8 9 10 Ethanol concentration (v/v%)
FIGURE 3
Bacterial growth (closed circle), consumed glycerol (open circle, dotted line), and ethanol concentration (open circle) after 24 hours cultivation of strain TB83. Bacterial growth corresponds to the second longitudinal axis. Ethanol concentration was calculated from the following equation: Actual ethanol concentration (%) = ethanol amount after 24 hours cultivation (%) initial ethanol amount (%).
medium (pH 9.0) containing 1.0, 2.0, 5.0, and 10.0% (v/v) ethanol. As shown in Fig. 3, ethanol productivity decreased by 40% with the addition of 2.0% (v/v) ethanol. With the addition of 5.0% (v/v) ethanol, no bacterial growth was observed and ethanol was not produced. This result shows that ethanol production of strain TB83 was inhibited by ethanol itself. Ethanol productivities by the mutants of other glycerol-assimilating ethanol producers exceed 20 g/L [19,20]. However, the ethanol-tolerance of these strains has not been clarified. In a single case, E. coli strains LY01, LY02, and LY03 mutated toward increased ethanol-tolerance, and produced more ethanol than did the wild strain [35]. This increased ethanol production may be due to improvement of ethanol-tolerance by the mutation. Molecular breeding by selecting for natural mutations is in progress to obtain other high ethanol producers and high ethanol-tolerant strains.
Conclusions
On the one hand, the addition of KCl showed no difference in comparison to the effect of NaCl (Table 5). Gonzalez et al. reported that bacterial growth and glycerol fermentation were inhibited in the presence of potassium ions [32]. However, bacterial growth was inhibited by the addition of 671 mM KCl, and ethanol production of strain TB-83 was not affected at less than 402 mM KCl. In both cases, although ethanol productivity slightly decreased from 20 g/ L of NaCl and KCl onwards, the molar yield finally increased. Since BDF waste that diluted 20 times with distilled water was used, salt concentration containing the media was be estimated to be at less than 10 g/L. These results indicate that ethanol production is not barely affected by salts such as NaCl and KCl at less than 20 g/L.
In this study, we obtained a novel high potential strain TB-83 that showed maximum ethanol productivity at alkaline pH. In addition, waste cooking oil-derived BDF, bBDF, facilitated higher ethanol production compared to wBDF, which is a new frying oil-derived BDF. Investigation of culture conditions demonstrated that the addition of yeast extract was highly effective in producing ethanol. However, the use of yeast extract in ethanol production from BDF waste is difficult to economically in terms of production costs, therefore, we need to examine the investigation of media component considering the production costs, in addition to the further improvement in ethanol production. To overcome this problem, we have been currently studying further investigation of a media component for the reduction of production costs. In addition, a further improved ethanol-tolerant strain obtained by mutation and scaled-up culture by pH control will be required, to further improve ethanol production.
Ethanol-tolerance
Acknowledgments
In ethanol production, it has been reported that low ethanol tolerance of bacteria is one of the major limiting factors for ethanol production [33,34]. Therefore, it is possible that ethanol production of strain TB-83 is affected by the ethanol concentration in the culture. To evaluate the ethanol tolerance of strain TB-83 at various ethanol concentrations, we used 1% glycerol basal
We thank Dr Masayuki Onodera (Niigata Institute of Technology) and Nagasaki Prefectural Institute for Environmental Research and Public Health for providing BDF waste. This work was supported by the Environment Research and Technology Development Fund (ERTDF) (3K123007) from the Ministry of the Environment, Government of Japan.
References [1] Ma F, Hanna MA. Biodiesel production: a review. Bioresour Technol 1999;70:1– 15. [2] He H, Wang T, Zhu S. Continuous production of biodiesel fuel from vegetable oil using supercritical methanol process. Fuel 2007;86:442–7. [3] Sakai S, Yagishita T. Microbial production of hydrogen and ethanol from glycerolcontaining wastes discharged from a biodiesel fuel production plant in a bioelectrochemical reactor with thionine. Biotechnol Bioeng 2007;98:340–8. [4] Ranganathan SV, Narasimhan SL, Muthukumar K. An overview of enzymatic production of biodiesel. Bioresour Technol 2008;99:3975–81. [5] Fukuda H, Kondo A, Noda H. Biodiesel fuel production by transesterification of oils. J Biosci Bioeng 2001;92:405–16. [6] Johnson DT, Taconi KA. The glycerin glut: options for the value-added conversion of crude glycerol resulting from biodiesel production. Environ Prog 2007;26:338–48. [7] da Silva GP, Mack M, Contiero J. Glycerol: a promising and abundant carbon source for industrial microbiology. Biotechnol Adv 2009;27:30–9. [8] Cheng KK, Liu HJ, Liu DH. Multiple growth inhibition of Klebsiella pneumoniae in 1,3-propanediol fermentation. Biotechnol Lett 2005;27:19–22.
252
www.elsevier.com/locate/nbt
[9] Mu Y, Teng H, Zhang DJ, Wang W, Xiu ZL. Microbial production of 1,3propanediol by Klebsiella pneumoniae using crude glycerol from biodiesel preparations. Biotechnol Lett 2006;28:1124–35. [10] Biebl H. Glycerol fermentation of 1,3-propanediol by Clostridium butyricum measurement of product inhibition by use of pH-auxostat. Appl Microbiol Biotechnol 1991;35:701–5. [11] Gonza´lez-Pajuelo M, Meynial-Salles I, Mendes F, Soucaille P, Vasconcelos I. Microbial conversion of glycerol to 1,3-propanediol: physiological comparison of a natural producer, Clostridium butyricum VPI 3266, and an engineered strain, Clostridium acetobutylicum DG1(pSPD5). Appl Environ Microbiol 2006;72:96–101. [12] Ito T, Nakashimada Y, Kakizono T, Nishio N. High-yield production of hydrogen by Enterobacter aerogenes mutants with decreased alpha-acetolactate synthase activity. J Biosci Bioeng 2004;97:27–232. [13] Ito T, Nakashimada Y, Senba K, Matsui T, Nishio N. Hydrogen and ethanol production from glycerol-containing wastes discharged after biodiesel manufacturing process. J Biosci Bioeng 2005;100:260–5. ¨ A, Santala V, Karp M. Hydrogen production from glycerol using halo[14] Kivisto philic fermentative bacteria. Bioresour Technol 2010;101:8671–7.
[15] Nwachukwu R, Shahbazi A, Wang L, Ibrahim S, Worku M, Schimmel K. Bioconversion of glycerol to ethanol by a mutant Enterobacter aerogenes. AMB Express 2012;2:20. [16] Jarvis GN, Moore ER, Thiele JH. Formate and ethanol are the major products of glycerol fermentation produced by a Klebsiella planticola strain isolated from red deer. J Appl Microbiol 1997;83:166–74. [17] Taconi KA, Venkataramanan KP, Johnson DT. Growth and solvent production by Clostridium pasteurianum ATCC 6013 utilizing biodiesel-derived crude glycerol as the sole carbon source. Environ Prog Sust Energy 2009;28:100–10. [18] Lee SJ, Kim SB, Kang SW, Han SO, Park C, Kim SW. Effect of crude glycerolderived inhibitors on ethanol production by Enterobacter aerogenes. Bioprocess Biosyst Eng 2012;35:85–92. [19] Oh BR, Seo JW, Heo SY, Hong WK, Luo LH, Joe MH, et al. Efficient production of ethanol from crude glycerol by a Klebsiella pneumoniae mutant strain. Bioresour Technol 2011;102:3918–22. [20] Saisaard K, Angelidaki I, Prasertsan P. Micro-aerobic, anaerobic and two-stage condition for ethanol production by Enterobacter aerogenes from biodieselderived crude glycerol. World Acad Sci Eng Technol 2011;53:795–8. [21] Choi WJ, Hartono MR, Chan WH, Yeo SS. Ethanol production from biodieselderived crude glycerol by newly isolated Kluyvera cryocrescens. Appl Microbiol Biotechnol 2011;89:1255–64. [22] Ban K, Kaieda M, Matsumoto T, Kondo A, Fukuda H. Whole cell biocatalyst for biodiesel fuel production utilizing Rhizopus oryzae cells immobilized within biomass support particles. Biochem Eng J 2001;8:39–43. [23] Sambrook J, Russell DW. Molecular cloning: a laboratory manual, 3rd ed., Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 2001. [24] Lane DJ. 16S/23S rRNA sequencing. In: Stackebrandt E, Goodfellow M, editors. Nucleic acid techniques in bacterial systematics. New York: Wiley; 1991. p. 115–47. [25] Tong I, Cameron D. Enhancement of 1,3-propanediol production by cofermentation in Escherichia coli expressing genes from Klebsiella pneumoniae dha regulon genes. Appl Biochem Biotechnol 1992;34/35:149–59.
RESEARCH PAPER
[26] Durnin G, Clomburg J, Yeates Z, Alvarez PJJ, Zygourakis K, Campbell P, et al. Understanding and harnessing the microaerobic metabolism of glycerol in Escherichia coli. Biotechnol Bioeng 2009;103:148–61. [27] Rosenblueth M, Martinez L, Silva J, Martinez-Romero E. Klebsiella variicola, a novel species with clinical and plant-associated isolates. Syst Appl Microbiol 2004;27:27–35. [28] Brisse S, Grimont F, Grimont PAD. The genus Klebsiella. Prokaryotes 2006;6:159– 96. [29] Nwachukwu RE, Shahbazi A, Wang L, Worku M, Ibrahim S, Schimmel K. Optimization of cultural conditions for conversion of glycerol to ethanol by Enterobacter aerogenes S012. AMB Express 2013;3:12. [30] Ashok S, Raj SM, Rathnasingh C, Park S. Development of recombinant Klebsiella pneumoniae DdhaT strain for the co-production of 3-hydroxypropionic acid and 1,3-propanediol from glycerol. Appl Microbiol Biotechnol 2011;90: 1253–65. [31] Murarka A, Dharmadi Y, Yazdani SS, Gonzalez R. Fermentative utilization of glycerol in Escherichia coli and its implications for the production of reduced chemicals and fuels. Appl Environ Microbiol 2008;74:1124–35. [32] Gonzalez R, Murarka A, Dharmadi Y, Yazdani SS. A new model for the anaerobic fermentation of glycerol in enteric bacteria: trunk and auxiliary pathways in Escherichia coli. Metab Eng 2008;10:234–45. [33] Sudha Rani K, Seenayya G. High ethanol tolerance of new isolates of Clostridium thermocellum strains SS21 and SS22. World J Microbiol Biotechnol 1999;15:173– 8. [34] Georgieva TI, Mikkelsen MJ, Ahring BK. High ethanol tolerance of the thermophilic anaerobic ethanol producer Thermoanaerobacter BG1L1. Cent Eur J Biol 2007;2:364–77. [35] Yomano LP, York SW, Ingram LO. Isolation and characterization of ethanol tolerant mutants of Escherichia coli KO11 for fuel ethanol production. J Ind Microbiol Biotechnol 1998;20:32–138.
www.elsevier.com/locate/nbt
253
Research Paper
New Biotechnology Volume 31, Number 3 May 2014
