- Email: [email protected]
Bioethanol fermentation by recombinant E. coli FBR5 and its robust mutant FBHW using hot-water wood extract hydrolyzate as substrate
Biotechnology Advances 28 (2010) 602–608
Contents lists available at ScienceDirect
Biotechnology Advances j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b i o t e c h a d v
Bioethanol fermentation by recombinant E. coli FBR5 and its robust mutant FBHW using hot-water wood extract hydrolyzate as substrate Tingjun Liu a,b, Lu Lin a,⁎, Zhijie Sun b, Ruofei Hu a,b, Shijie Liu a,b,⁎ a b
State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou, Guangdong 510641, PR China Department of Paper and Bioprocess Engineering, State University of New York – College of Environmental Science and Forestry, Syracuse, NY 13210, USA
a r t i c l e
i n f o
Available online 15 May 2010 Keywords: E. coli FBR5 Strain adaptation Xylose Ethanol fermentation Hemicellulose Dilute acid hydrolysis
a b s t r a c t Hemicellulose is a potential by-product currently under-utilized in the papermaking industry. It is a heterocarbohydrate polymer. For hardwood hemicelluloses, D-xylose is the major component upon depolymerization. At SUNY-ESF, wood extracts were obtained by extracting sugar maple wood chips with hot water at an elevated temperature. The wood extracts were then concentrated and acid hydrolyzed. Ethanologenic bacteria, E. coli FBR5, had a good performance in pure xylose medium for ethanol production. However, FBR5 was strongly inhibited in dilute sulfuric acid hydrolyzate of hot-water wood extract. FBR5 was challenged by hot-water wood extract hydrolyzate in this study. After repeated strain adaptation, an improved strain: E. coli FBHW was obtained. Fermentation experiments indicated that FBHW was resistant to the toxicity of hydrolyzate in the fermentation media of concentrated hydrolyzate, and xylose was completely utilized by the strain to produce ethanol. FBHW was grown in the concentrated hydrolyzate without any detoxification treatment and has yielded 36.8 g/L ethanol. © 2010 Elsevier Inc. All rights reserved.
1. Introduction Since the 1970s, renewable fuels and materials have attracted significant interest due to high petroleum prices and awareness of the depletion of fossil fuel reserves. Life on earth ultimately depends on photosynthesis, which results in the production of plant biomass with cellulose as its main component. Plant biomass is the only foreseeable sustainable resource of fuels and materials available to humanity (Lee et al., 2002). Bioethanol production is considered to be a milestone for sustainable development. Hydrolyzate of biomass includes a number of pentoses and hexoses. There is no single organism yet shown to efficiently convert all these sugars to ethanol (Nan and Paul, 2008). In the last two decades, numerous microorganisms have been engineered to selectively produce ethanol. The greatest successes have been in the engineering of Gram-negative bacteria: Escherichia coli, Klebsiella oxytoca, and Zymomonas mobilis. E. coli and K. oxytoca are naturally able to use a wide spectrum of sugars; Z. mobilis produces ethanol at high yields, but ferments only glucose and fructose. Work has concentrated on engineering E. coli and K. oxytoca to selectively produce ethanol and introducing pathways into Z. mobilis for the fermentation of arabinose and xylose (Dien et al. 2003). Dien et al. produced an improved xylose-fermenting strain (E. coli FBR5) based on the host NZN111 (Qureshi et al., 2006; Dien et al., ⁎ Corresponding authors. State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou, Guangdong 510641, PR China. E-mail addresses: [email protected] (L. Lin), [email protected] (S. Liu). 0734-9750/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.biotechadv.2010.05.008
2000). Transformation was accomplished with the plasmid pLOI297 bearing the PET (production of ethanol) operon developed earlier by Ingram and colleagues (Ingram et al., 1987; Beall et al., 1991). The plasmid pLOI297 consists of the pyruvate decarboxylase (pdc) and alcohol dehydrogenase genes (adh) from Zymomonas mobilis and genes conferring resistance to tetracycline. In anaerobic batch cultures, strain FBR5 was shown to produce ethanol in high yields on glucose and xylose (Martin Gregory et al., 2006). Hemicellulose is a potential by-product currently under-utilized in the papermaking industry. It is a hetero-carbohydrate polymer, which can be hydrolyzed into monosaccharides by a dilute acid process. In addition to aromatics, acetic acid, formic acid and methanol released from woody biomass during the process, the hydrolysis of carbohydrates also generates toxic compounds. During dilute acid hydrolysis of biomass, monosaccharides are dehydrated and various toxic compounds are formed that can inhibit microorganism growth and ethanol fermentation, such as furfural and 5-hydroxymethylfurfural (HMF) (Delgenes et al., 1996; Modig et al., 2002). Lignin byproducts during biomass hydrolysis are also inhibitory to microorganisms. This mixture of inhibitors will likely be a great challenge to these fermenting strains. Many methods for detoxification have been investigated, including neutralization, overliming, evaporation, the use of ion-exchange resins, and activated charcoal adsorption (Ranatunga et al., 1997; Carvalho et al., 2004). Strain adaptation has also been successful by addition of furfural and ethanol into media and is as shown in a pending US patent (Lefebvre et al., 2007, 2008). Sugar Maple hot-water extract hemicellulosic hydrolyzate was concentrated and fractionated by a Nano-filtration membrane process
T. Liu et al. / Biotechnology Advances 28 (2010) 602–608
in our lab. In this study, E. coli FBR5 was challenged by hot-water wood extract hemicellulosic hydrolyzate. After repeated strain adaptation, a new improved strain: E. coli FBHW was obtained. Fermentation experiments indicated that FBHW was resistant to the toxicity of hydrolyzate in a fermentation medium of concentrated hydrolyzate, and that xylose was completely utilized by the strain to produce ethanol. FBHW was grown in concentrated hydrolyzate without any detoxification treatment and yielded 36.8 g/L ethanol. 2. Materials and methods 2.1. Processing of hemicellulose hydrolyzate from sugar maple Sugar maple wood chips were extracted by hot water at about 160 °C for 2 h. The hot-water extraction was carried out in a 65 ft3 digester with a wood to liquor ratio about 1:4. The wood extract was concentrated about 10-fold by a Nano-filtration membrane. The concentrated wood extract was hydrolyzed at the temperature of 135 °C for 25 min with 1% (wt.%, [H+] = 0.204 mol/L as catalyst) sulfuric acid added. Acid-insoluble lignin was centrifuged out (CEPA High speed centrifuge Z81G, cylinder speed 16,000 rpm, cylinder diameter 125 mm, New Brunswick Scientific, NJ 08818-4005, USA). Hydrolyzate was neutralized by Ca(OH)2 and NaOH, and then fractionated by Nano-filtration. Two mixtures of acids were also tested for hydrolysis of concentrated hot-water wood extract. In order to maintain [H+] = 0.204 mol/L as catalyst, a carefully prescribed mixture of three acids (0.33% H2SO4 + 0.43% HNO3 + 0.25% HCl, wt.%) was added into the concentrated wood extract and temperature kept at 135 °C for 10 min. Hydrolyzate was then neutralized by a mixture of Ca(OH)2 (for 0.33% H2SO4) and NaOH (for 0.43% HNO3 + 0.25% HCl). Another batch of concentrated hot-water wood extract was hydrolyzed by a two acid mixture (0.5% H2SO4 + 0.65% HNO3, wt.%) at a temperature of 135 °C for 10 min, and then neutralized by Ca(OH)2. 2.2. Microorganism and cell growth E. coli FBR5 was maintained at 4 °C in modified Luria-Bertani (LB Broth, Lennox, Fisher Scientific, Fair Lawn, New Jersey, USA) agar solid medium containing 10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl and 15 g/L agar, adjusted to a pH of 7.0, supplemented with 4 g/L xylose
603
(MP Biomedicals, Solon, Ohio, USA) and 20 mg/L tetracycline (MP Biomedicals, Solon, Ohio, USA). In order to prepare inocula, 2 loops of cells were transferred to 125 mL screw capped plastic flasks (NALGENE, Rochester, NY, USA) containing 50 mL sterile liquid medium. The liquid medium contained 10 g/L tryptone, 5 g/L yeast extract and 5 g/L NaCl, adjusted to a pH of 7.0, supplemented with 10 g/L xylose and 20 mg/L tetracycline. LB broth and xylose stock solution (400 g/L) were autoclaved (HIRAYAMA HICLAVETM HV-110, Amerex Instruments, Lafayette, CA, USA) separately at 121 °C for 15 min, followed by cooling to room temperature (approximately 45 °C for the LB agar solid medium) and then mixed. Filter-sterilized (0.22 µm sterile filter, NALGENE, Rochester, NY, USA) tetracycline stock solutions (20 mg/mL) were added to the above concentrations. The liquid culture was incubated at 35 °C for 12–14 h on a rotary shaker (GYROMAXTM 747R, Amerex Instruments, Lafayette, CA, USA). Shake speed was set at 160 rpm. After growth, 5 mL of culture were used to inoculate 100 mL of sterile fermentation medium. 2.3. Strain adaptation and fermentation in flask Original E. coli FBR5 was maintained at 4 °C on modified LB plate. In order to prepare active cells, a colony was transferred to 50 mL sterile liquid medium and incubated overnight at 35 °C. Strain adaptation was performed by exposing an FBR5 overnight culture to fermentation medium containing 10% (v/v) concentrated hydrolyzate and plating out dilutions on selective medium containing 10% (v/v) hydrolyzate. The selective medium contained the same ingredients as the solid medium but replaced xylose with concentrated hot-water wood extract hydrolyzate at different levels. The largest colonies were selected and plated on selective medium of the same concentrated hydrolyzate level in comparison with the parent. Inoculated Petri dishes were incubated at 35 °C (IncuMax IC150, Amerex Instruments, Lafayette, CA, USA). Colonies bigger than the parent were selected for fermentation test in a fermentation medium containing 20% (v/v) concentrated hydrolyzate. The strain of highest ethanol production was selected for dilution and plating on selective medium containing 20% (v/v) concentrated hydrolyzate. The selected strain was passed through concentrated hydrolyzate challenges several times while increasing hydrolyzate concentration gradually from 10% to 20%, 30%, 40% and 50%. The final strain obtained after successive adaptation, E. coli FBHW, was stored and used in the subsequent fermentation study.
Fig. 1. 600 MHz 1H-NMR spectrum for the concentrated hot-water wood extract hydrolyzate before membrane separation.
604
T. Liu et al. / Biotechnology Advances 28 (2010) 602–608
Fig. 2. 600 MHz 1H-NMR spectrum for the concentrated hot-water wood extract hydrolyzate after membrane separation.
Fermentations were performed in 125 mL screw capped plastic flasks containing 100 mL sterile fermentation medium at 35 °C for 96–144 h on a rotary shaker (shake speed set at 160 rpm). Samples were taken routinely until fermentation stopped. The fermentation medium contained the same ingredients as the liquid medium for inocula but replaced xylose with concentrated hot-water wood extract hydrolyzate at different levels. 2.4. Ion-exchange treatment of hydrolyzate C-100H, strong acid cation exchange resin, and A-400, strong base anion exchange resin (Purolite, Bala Cynwyd, PA, USA), were used for desaltification of dilute sulfuric acid wood extract hydrolyzate. The process of desaltification was carried out in 250 mL chromatographic column. After ion-exchange, hydrolyzate was adjusted to pH7.0 with Ca(OH)2 for fermentation study. 2.5. Fermentation in bioreactor Fermentations with pH control were carried out in a 1 L New Brunswick Bioreactor (BIOFLO 110; New Brunswick Scientific Co., New Brunswick, NJ, USA). Fermentation pH was controlled at 7.0 using 2.5 mol/L NaOH solution. The working volume of the bioreactor was 800 mL. LB Broth (16 g) (Lennox, Fisher Bioreagents, Fair Lawn, NJ, USA) was dissolved in X mL of distilled water in the bioreactor. The bioreactor was autoclaved at 121 °C for 15 min followed by cooling it to room temperature. (800 − X) mL of concentrated hydrolyzate was sterilized separately by autoclaving at 121 °C for 15 min in a glass bottle or passing through 0.22 µm sterile filter (nitrocellulose membrane, Millipore) which was held on a filter holder with receiver (1000 mL, NALGENE, Rochester, NY, USA). Different sugar concentrations of fermentation media were achieved by changing the values of X. For example, 200, 280 and 360 mL of distilled water for the media containing 136, 111 and 96.7 g/L reducing sugar, respectively. After cooling, concentrated hydrolyzate was added to the bioreactor and adjusted to pH 7.0 prior to inoculation. The bioreactor was inoculated with 40 mL of actively growing 12–14 h-old culture and incubated at 35 °C. Agitation speed was set at 200 rpm and air flow rate was set as 25 ccm.
2.6. Analytical methods Cell density (g/L) was estimated by using a predetermined correlation between dry weight cell concentrations (oven dry at 105 °C) versus optical density. An OD of one is equivalent to 0.31 g/L of dry cells (data not shown). One mL of fermentation broth was centrifuged to harvest cells. Cells were then washed twice with 1 mL 0.9% NaCl solution to get rid of color from the hydrolyzate. Optical density was measured at 540 nm after suspending the cells with 1 mL 0.9% NaCl solution. Cell numbers in the fermentation broth were counted under a microscope (Model: 12-575-251, Fisher Scientific, Fair Lawn, New Jersey, USA) (Shuler and Kargi, 2001). Ethanol concentration was measured by GC using Thermo Scientific Focus GC systems equipped with a Triplus automatic sampler and a TRACE TR-WaxMS (30 m × 0.25 mm × 0.25 µm) GC column. The GC oven was set to an initial temperature of 50 °C and held for 3 min, after which the temperature was increased at a rate of 30 °C per min until it reached a final temperature of 180 °C. The injector temperature was set as 200 °C and run on split mode with a constant flow of 1.5 mL/min. Samples were prepared by adding 100 µL of sample and 100 µL of internal standard (t-butanol 5.5 g/L) with 800 µL of distilled water. Total reducing sugars were quantified using 3,5-dinitrosalicylic acid method (DNS method, ACROS, New Jersey, USA) (Hu et al., 2008). Xylose concentration was measured by enzymatic assay kit (Megazyme, D-Xylose assay kit, Bray, Co. Wicklow, Ireland, 2008). For ethanol and sugar analysis, samples (1 mL) were taken intermittently and clarified by centrifugation (Micro-centrifuge 235C, Fisher
Table 1 Inhibitors chemical shift and concentration from 600 MHz 1H-NMR spectra. Inhibitor
Chemical shift ppm
Peak area
Concentration, g/L
Before
After
Before
After
Acetic acid Formic acid HMF Furfural
2.1 8.25 9.4 9.5
6651.45 N/A 7.30 111.31
371.06 N/A N/A N/A
38.86 N/A 0.27 3.12
2.17 N/A N/A N/A
T. Liu et al. / Biotechnology Advances 28 (2010) 602–608
Fig. 3. E. coli FBR5 in concentrated hot-water wood extract hydrolyzate medium containing 59.1 g/L reducing sugar.
Scientific, Fair Lawn, New Jersey, USA) at 13,600 × g for 5 min to remove cells. Supernatants were stored in eppendorf tubes at − 20 °C and analyzed at the end of each batch fermentation. Fermentation inhibitors (acetic acid, formic acid, furfural and HMF) concentrations in the concentrated wood extract hydrolyzate were determined by 600 MHz 1H-NMR (Bruker BioSpin Corporation, Billerica, MA, USA). After centrifugation, samples were prepared by adding 100 µL of sample and 100 µL of internal standard (TSP, SigmaAldrich, St Louis, MO, USA) with 800 µL of distilled water. Samples were homogenized in a mixer. The results were calculated by the ratio of inhibitors and TSP's peak area (Kiemle et al., 2004; Stipanovic and Kiemle, 2002; Copur et al., 2003). The NMR operating conditions are as follows, Probe Type: Broadband Observe Probe; Temperature: 30 °C; 90° Pulse: 11 μs; Interval between pulses: 10 s; Time for acquisition: 2.73 s; Sweep Width: 10 ppm; Center of spectrum: 4.5 ppm; Reference: Acetone at 2.2 ppm. The area of each peak was integrated by MestReNova software and the integral of TSP was set as 100. The concentration of total phenols in the concentrated wood extract hydrolyzate was measured by 4-aminoantipyrine colorimetric method (Standard Methods Committee, 1988). Ion concentrations of the concentrated wood extract hydrolyzate were analyzed by inductively coupled plasma (ICP). Samples were analyzed on a Perkin Elmer 3300DV Inductively Coupled Plasma Emission Spectrometer. Samples were diluted 100-fold with 2% nitric acid for analysis. The element concentrations were obtained via emissions from the following lines (in nanometers): Fe — 238.204; Ca — 317.933; Na — 589.952; S — 181.975; Cr — 205.960; Cu — 324.752; Mo — 202.031; Ni — 231.604; K — 766.490; P — 178.221; Mg — 285.213. Ethanol productivity was calculated as the maximum ethanol produced (g/L) divided by the fermentation time (h) to achieve maximum ethanol concentration and is expressed in g/(L ⁎ h). Ethanol yield (YP/S) was calculated as the maximum ethanol produced (g) divided by total reducing sugar consumed (g).
605
Fig. 4. Sugar utilization during fermentation of untreated and desalted hydrolyzate.
3. Results and discussion 3.1. 600 MHz 1H-NMR analysis for the concentrated hot-water wood extract hydrolyzate before and after membrane separation The acetyl groups from hemicellulose contribute to acetic acid formation in the extraction liquor. Our experimental results show that the pH of extraction liquor drops from the initial neutral conditions (∼pH7.0) to acidic conditions (∼pH3.2). This acidic condition during the hot-water extraction process further catalyzes the extraction and hydrolysis reactions. The extraction proceeds slowly initially and faster as pH decreases during extraction. Therefore, the hot-water extraction reactions are referred to as autocatalysis, and sometimes as autohydrolysis. A major portion of hemicellulose was extracted in the form of soluble oligomers and monomers in the extraction liquor (Liu, 2008). Upon membrane processing, water and other low molecular weight compounds, e.g. acetic acid, formic acid, methanol, furfural, etc., were preferentially sent to the permeate stream, while sugar monomers and oligomers along with high molecular weight compounds were concentrated in the concentrate stream. Xylose and acetic acid are the largest components recovered from the hydrolysis process.
Table 2 Metal ion, inhibitor and sugar concentration of the hydrolyzate before and after ionexchange.
Untreated Treated
Na, ppm
S, ppm
Ca, ppm
Xylose, g/L
Reducing sugar, g/L
Phenol, mg/L
8548 520.3
4460 318.9
414 4948
37.5 34.5
111.6 105.3
438.4 288.0
Fig. 5. Ethanol production during fermentation of untreated and desalted hydrolyzate.
606
T. Liu et al. / Biotechnology Advances 28 (2010) 602–608
Table 3 Ion concentration (ppm) of the hydrolyzate catalyzed by different mixed acids.
2-mixed acids 3-mixed acids
Na
S
Ca
Fe
6 275
902 1422
6106 3800
1819 3300
In 600 MHz 1H-NMR spectrum, the peaks of acetic acid, formic acid, HMF and furfural appeared at 2.1, 8.25, 9.4 and 9.5 ppm, respectively. As can be seen in Fig. 1, the peak area of acetic acid is very high (6651.45 at 2.1 ppm). The concentration of acetic acid is 38.86 g/ L. After membrane separation, a significant amount of the inhibitors have been removed from the hydrolyzate. No formic acid, furfural and HMF are detected by 600 MHz 1H-NMR (Fig. 2). Total phenol content decreased from 940.8 mg/L to 404.8 mg/L after membrane filtration. Reducing sugar concentration was 212 g/L. The data on chemical shift and concentration of inhibitors in the concentrated hot-water wood extract hydrolyzate before and after membrane separation are summarized in Table 1. 3.2. Fermentation of concentrated hot-water wood extract hydrolyzate catalyzed by dilute sulfuric acid
Fig. 7. Fermentation results of concentrated (3-mixed-acid) hydrolyzate medium containing 134 g/L reducing sugar.
comparison with results shown in Fig. 3, this one shows half of the productivity and yield found for hydrolysate medium containing 59.1 g/L reducing sugar.
After repeated strain adaptation, FBR5's growth was slightly inhibited in the concentrated hydrolyzate medium containing 59.1 g/L total reducing sugar (15.2 g/L xylose, data not shown in the figure). In Fig. 3, one can observe that cells grew slowly in the first two days, and then reached the exponential growth phase in the third day, at which point xylose utilization rate decreased. During the first day, 0.2 g/L of xylose were utilized. It took 4 days to consume all the xylose and 29.2 g/L reducing sugar in the medium. Only 0.47 g/L ethanol was produced in the first day of fermentation. Ethanol concentration increased over the next few days, and a maximum concentration of 9.30 g/L was achieved on the fifth day. Ethanol productivity was 0.078 g/(L ⁎ h) with a yield (YP/S) of 0.32 g ethanol/g consumed reducing sugar. After strain adaptation, a mutant was isolated and named as E. coli FBHW. FBHW was used in subsequent fermentation studies. In further research, FBHW's growth and ethanol production were found to be significantly inhibited in more concentrated hot-water wood extract hydrolyzate medium containing 111.6 g/L reducing sugar (containing 37.5 g/L xylose). During fermentation over four days, 13.73 g/L of xylose and 23.3 g/L of reducing sugar was utilized and 3.49 g/L of ethanol was produced. Ethanol productivity was 0.036 g/(L ⁎ h) with a yield (YP/S) of 0.15 g ethanol/g consumed reducing sugar. In
According to the 600 MHz 1H-NMR result most of the fermentation inhibitors have been removed from the hydrolyzate, yet cell growth was found to be still inhibited. Na2SO4 was formed during neutralization of hydrolyzate, and it cannot pass through the nanofiltration membrane. To investigate inhibition effect of Na2SO4 on fermentation, the hydrolyzate was treated by an ion-exchange resin, then adjusted pH to 7.0 with Ca(OH)2 for fermentation studies. One can see from Table 2 that the concentrations of Na and S decreased after ion exchange treatment. The concentration of Ca increased, due to adjustment of pH with Ca(OH)2. Concentrations of xylose and reducing sugar all decreased slightly, from 37.5 and 111.6 g/L to 34.5 and 105.3 g/L, respectively. The loss rate of xylose and reducing sugar is 8% and 5.6%, respectively. Total phenol content also dropped from 438.4 to 288.0 mg/L. In Fig. 4 we can see that xylose and reducing sugar was utilized quickly and thoroughly by E. coli after ion-exchange treatment. In the fermentation of treated hydrolyzate 34.3 g/L of xylose was consumed after 72 h of fermentation, with 50.8 g/L of reducing sugar being
Fig. 6. Fermentation results of concentrated (2-mixed-acid) hydrolyzate medium containing 118 g/L reducing sugar.
Fig. 8. Fermentation results of concentrated (3-mixed-acid) hydrolyzate medium containing 111 g/L reducing sugar.
3.3. Fermentation of dilute sulfuric acid hydrolyzate after ion-exchange
T. Liu et al. / Biotechnology Advances 28 (2010) 602–608
Fig. 9. Fermentation results of autoclaved concentrated (3-mixed-acid) hydrolyzate media containing 136 or 96.7 g/L reducing sugar.
consumed. In the fermentation of hydrolyzate without ion-exchange treatment 13.7 g/L xylose was consumed after 96 h of fermentation, with 23.3 g/L of reducing sugar being consumed. From Fig. 5 we can see that the highest ethanol concentration was 22.6 g/L after 96 h (4 days) fermentation of the treated hydrolyzate. The ethanol productivity was 0.24 g/(L ⁎ h) with a yield (YP/S) of 0.45 g ethanol/g consumed reducing sugar. In the fermentation of untreated hydrolyzate 3.49 g/L ethanol was produced after 96 h of fermentation. The ethanol productivity for untreated hydrolyzate was 0.036 g/(L ⁎ h) with a yield (YP/S) of 0.15 g ethanol/g consumed reducing sugar. 3.4. Fermentation of hydrolyzate catalyzed by dilute mixed acids Table 3 shows the concentration of Na, S, Ca and Fe. Fig. 6 shows that the highest ethanol concentration of 2.62 g/L was achieved at the second day of fermentation, and then decreased during the next 2 days of fermentation. The high concentration of Ca may be a cause of cell growth and ethanol production inhibition. Only 2.34 g/L of ethanol was produced and only 15 g/L of reducing sugar was consumed after 96 h of fermentation. The ethanol productivity was 0.055 g/(L⋅h) with
607
a yield (YP/S) of 0.26 g ethanol/g consumed reducing sugar after 48 h of fermentation. Strain adaptation is continuing. The last results reported here are shown in Fig. 7. The hydrolyzate produced from a 3-mixed acid hydrolysis was concentrated to 134 g/L of reducing sugar by nanofiltration membrane and used as the fermentation substrate, directly without any detoxification pretreatment. As can be seen in Fig. 7, sugar concentration decreased gradually, and 108 g/L reducing sugar was utilized after 96 h of fermentation. The maximum ethanol concentration of 36.9 g/L was achieved at the end of 96 h of fermentation. Ethanol productivity was 0.38 g/(L ⁎ h) with a yield (YP/S) of 0.34 g ethanol/g consumed reducing sugar. The extract hydrolyzate at 134 g/L reducing was diluted with distilled water to 111 g/L reducing sugar and the fermentation was tested again. Higher ethanol productivity was achieved by using this less concentrated hydrolyzate as substrate (Fig. 8), most likely due to lower concentrations of sugar and inhibitors. When the reducing sugar content of the hydrolyzate medium was adjusted to 111 g/L, 34.8 g/L of ethanol was produced and 90.1 g/L of reducing sugar was consumed during 72 h of fermentation. The ethanol productivity was 0.48 g/(L ⁎ h) with a yield (YP/S) of 0.39 g ethanol/g consumed reducing sugar. 3.5. Comparison of sterilization methods for hydrolyzate The effect of different hydrolyzate sterilization methods on cell growth and ethanol production was investigated. From Fig. 9 we can see that E. coli utilized sugar and produced ethanol slowly in media containing hydrolyzate that had been autoclaved. In the fermentation of hydrolyzate medium containing 136 g/L reducing sugar, 73 g/L reducing sugar was consumed after 144 h of fermentation. Ethanol concentration increased slowly, with only 0.27 g/L ethanol produced after 48 h of fermentation. The maximum ethanol concentration of 20.1 g/L was achieved after 96 h of fermentation and decreased thereafter. The ethanol productivity was 0.21 g/(L ⁎ h) with a yield (YP/S) of 0.34 g ethanol/g consumed reducing sugar. In the fermentation of hydrolyzate medium containing 96.7 g/L reducing sugar, the maximum ethanol concentration of 32.0 g/L was achieved after 96 h of fermentation, with 78.5 g/L reducing sugar being consumed. The ethanol productivity was 0.33 g/(L ⁎ h) with a yield (YP/S) of 0.41 g ethanol/g consumed reducing sugar. Fig. 10, in comparison with Fig. 2,
Fig. 10. 600 MHz 1H-NMR spectrum for the concentrated hot-water wood extract (3-mixed-acid) hydrolyzate after autoclaving.
608
T. Liu et al. / Biotechnology Advances 28 (2010) 602–608
shows that acetic acid concentration increased while polymer acetyl group decreased upon autoclaving. A small amount of furfural and sediment was formed during hydrolyzate autoclaving. The concentration of acetic acid and furfural was 2.63 and 0.25 g/L, respectively, in the autoclaved hydrolyzate containing 212 g/L reducing sugar. 4. Concluding remarks In conclusion, E. coli FBR5 can be adapted to hot-water wood extract hydrolyzate (containing 59.1 g/L reducing sugar) catalyzed by dilute sulfuric acid and can utilize xylose completely after strain adaptation; however, E. coli's growth is still strongly inhibited in more concentrated hydrolyzate containing 111.6 g/L reducing sugar. Inhibition is decreased after ion-exchange. In the fermentation of hydrolyzate catalyzed by 3-mixed-acids, cells grow fast and produce 36.9 g/L ethanol after 96 h of culture. The hydrolyzate catalyzed by 2-mixed-acids shows significant inhibition on cell growth and ethanol production. The concentration of acetic acid and furfural is increased during hydrolyzate autoclaving. The autoclaved hydrolyzate exhibits some increased inhibition of cell growth and ethanol production. Acknowledgements The authors are indebted to Dr. Bruce Dien for providing the Escherichia coli FBR5 samples to carry out this study. USDOE, NYSERDA and China National Key Research Program (2010CB732201) are gratefully acknowledged for their financial supports. The authors are grateful for the staff in the Bioprocess Engineering lab, SUNY ESF for their support in completing this research. Dr. Thomas E. Amidon is especially acknowledged for his support in getting this work done. We would also like to thank Mr. Dave Kiemle, Mr. Christopher D. Wood and Ms. Debra A. Driscoll for their help with these studies. References Beall DS, Ohta K, Ingram LO. Parametric studies of ethanol production from xylose and other sugars by recombinant Escherichia coli. Biotechnol Bioeng 1991;38:296–303. Carvalho Wde, Canilha L, Mussatto SI, Dragone G, Morales MLV, Solenzal AIN. Detoxification of sugarcane bagasse hemicellulosic hydrolysate with ion-exchange
resins for xylitol production by calcium alginate-entrapped cells. J Chem Technol Biotechnol 2004;79:863–8. Copur Y, Kiemle DJ, Stipanovic A, Koskinen J, Makkonen H. 1H-NMR spectroscopic determination of carbohydrates and yield in pine and maple pulps. Paperi ja Puu 2003;85:158–62. Delgenes JP, Moletta R, Navarro JM. Effects of lignocellulose degradation products on ethanol fermentations of glucose and xylose by Saccharomyces cerevisae, Zymomonas mobilis, Pichia stipitis, and Candida shehatae. Enzyme Microb Technol 1996;19:220–5. Dien BS, Nichols NN, O'Bryan PJ, Bothast RJ. Development of new ethanologenic Escherichia coli strains for fermentation of lignocellulosic biomass. Appl Biochem Biotechnol 2000;84:181–96. Dien BS, Cotta MA, Jeffries TW. Bacteria engineered for fuel ethanol production: current status. Appl Microbiol Biotechnol 2003;63:258–66. Hu RF, Lin L, Liu TJ, Liu SJ. Reducing sugar content in hemicellulose hydrolysate by DNS method: a revisit. J Biobased Mater Bioeng 2008;2(2):156–61. Ingram LO, Conway T, Clark DP, Sewell GW, Preston JF. Genetic engineering of ethanol production in Escherichia coli. Appl Environ Microbiol 1987;53(10):2420–5. Kiemle DJ, Stipanovic AJ, Mayo KE. Proton NMR methods in the compositional characterization of polysaccharides. ACS Symp series 2004;864:122–39. Lee RL, Paul JW, Van Z. Willem H,, Isak SP, Microbial Cellulose Utilization: Fundamentals Biotechnology. Microbiol Molec Biology Rev 2002;66(3):506–77. Lefebvre BG, Savelski MJ, Hecht GB. Ethanologenic bacteria, International application number: PCT/US2007/021892, 12 October 2007. Lefebvre BG, Savelski MJ, Hecht GB. Ethanol resistant and furfural resistant strains of E. coli FBR5 for production of ethanol from cellulosic biomass, International publication number: WO 2008/048513 A2, 24 April 2008. Liu SJ. A kinetic model on autocatalytic reactions in woody biomass hydrolysis. J Biobased Mater Bioeng 2008;2(2):135–47. Nan Fu, Paul P. Co-fermentation of a mixture of glucose and xylose to ethanol by Zymomonas mobilis and Pachysolen tannophilus. World J Microbiol Biotechnol 2008;24:1091–7. Martin Gregory JO, Knepper A, Zhou B, Pamment NB. Performance and stability of ethanologenic Escherichia coli strain FBR5 during continuous culture on xylose and glucose. J Ind Microbiol Biotechnol 2006;33:834–44. Megazyme, Bray, Co. Wicklow, Ireland, 2008; Website: http://secure.megazyme.com. Modig T, Liden G, Taherzadeh MJ. Inhibition effects of furfural on alcohol dehydrogenase, aldehyde dehydrogenase, and pyruvate dehydrogenase. Biochem J 2002;363:769–76. Qureshi N, Dien BS, Nichols NN, Saha BC, Cotta MA. Genetically engineered Escherichia coli for ethanol production from xylose: substrate and product inhibition and kinetic parameters. Inst Chem Eng: Food Bioproducts Process 2006;84(C2):114–22. Ranatunga TD, Jervis J, Helm RF, McMillan JD, Hatzis C. Toxicity of hard-wood extractives toward Saccharomyces cerevisiae glucose fermentation. Biotechnol Lett 1997;19:1125–7. Shuler ML, Kargi F. Bioprocess Engineering: Basic Concepts. Second Edition. NJ, USA: Prentice Hall; 2001. p. 156. Standard Methods Committee. 5530 Phenols. Aggregate organic constituents; 1988. 5000:5/48. Stipanovic A, Kiemle DJ. Proton NMR methods in the compositional characterization of hemicelluloses. Abstracts of Papers, 223rd ACS National Meeting; 2002. April 7–11.
Contents lists available at ScienceDirect
Biotechnology Advances j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b i o t e c h a d v
Bioethanol fermentation by recombinant E. coli FBR5 and its robust mutant FBHW using hot-water wood extract hydrolyzate as substrate Tingjun Liu a,b, Lu Lin a,⁎, Zhijie Sun b, Ruofei Hu a,b, Shijie Liu a,b,⁎ a b
State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou, Guangdong 510641, PR China Department of Paper and Bioprocess Engineering, State University of New York – College of Environmental Science and Forestry, Syracuse, NY 13210, USA
a r t i c l e
i n f o
Available online 15 May 2010 Keywords: E. coli FBR5 Strain adaptation Xylose Ethanol fermentation Hemicellulose Dilute acid hydrolysis
a b s t r a c t Hemicellulose is a potential by-product currently under-utilized in the papermaking industry. It is a heterocarbohydrate polymer. For hardwood hemicelluloses, D-xylose is the major component upon depolymerization. At SUNY-ESF, wood extracts were obtained by extracting sugar maple wood chips with hot water at an elevated temperature. The wood extracts were then concentrated and acid hydrolyzed. Ethanologenic bacteria, E. coli FBR5, had a good performance in pure xylose medium for ethanol production. However, FBR5 was strongly inhibited in dilute sulfuric acid hydrolyzate of hot-water wood extract. FBR5 was challenged by hot-water wood extract hydrolyzate in this study. After repeated strain adaptation, an improved strain: E. coli FBHW was obtained. Fermentation experiments indicated that FBHW was resistant to the toxicity of hydrolyzate in the fermentation media of concentrated hydrolyzate, and xylose was completely utilized by the strain to produce ethanol. FBHW was grown in the concentrated hydrolyzate without any detoxification treatment and has yielded 36.8 g/L ethanol. © 2010 Elsevier Inc. All rights reserved.
1. Introduction Since the 1970s, renewable fuels and materials have attracted significant interest due to high petroleum prices and awareness of the depletion of fossil fuel reserves. Life on earth ultimately depends on photosynthesis, which results in the production of plant biomass with cellulose as its main component. Plant biomass is the only foreseeable sustainable resource of fuels and materials available to humanity (Lee et al., 2002). Bioethanol production is considered to be a milestone for sustainable development. Hydrolyzate of biomass includes a number of pentoses and hexoses. There is no single organism yet shown to efficiently convert all these sugars to ethanol (Nan and Paul, 2008). In the last two decades, numerous microorganisms have been engineered to selectively produce ethanol. The greatest successes have been in the engineering of Gram-negative bacteria: Escherichia coli, Klebsiella oxytoca, and Zymomonas mobilis. E. coli and K. oxytoca are naturally able to use a wide spectrum of sugars; Z. mobilis produces ethanol at high yields, but ferments only glucose and fructose. Work has concentrated on engineering E. coli and K. oxytoca to selectively produce ethanol and introducing pathways into Z. mobilis for the fermentation of arabinose and xylose (Dien et al. 2003). Dien et al. produced an improved xylose-fermenting strain (E. coli FBR5) based on the host NZN111 (Qureshi et al., 2006; Dien et al., ⁎ Corresponding authors. State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou, Guangdong 510641, PR China. E-mail addresses: [email protected] (L. Lin), [email protected] (S. Liu). 0734-9750/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.biotechadv.2010.05.008
2000). Transformation was accomplished with the plasmid pLOI297 bearing the PET (production of ethanol) operon developed earlier by Ingram and colleagues (Ingram et al., 1987; Beall et al., 1991). The plasmid pLOI297 consists of the pyruvate decarboxylase (pdc) and alcohol dehydrogenase genes (adh) from Zymomonas mobilis and genes conferring resistance to tetracycline. In anaerobic batch cultures, strain FBR5 was shown to produce ethanol in high yields on glucose and xylose (Martin Gregory et al., 2006). Hemicellulose is a potential by-product currently under-utilized in the papermaking industry. It is a hetero-carbohydrate polymer, which can be hydrolyzed into monosaccharides by a dilute acid process. In addition to aromatics, acetic acid, formic acid and methanol released from woody biomass during the process, the hydrolysis of carbohydrates also generates toxic compounds. During dilute acid hydrolysis of biomass, monosaccharides are dehydrated and various toxic compounds are formed that can inhibit microorganism growth and ethanol fermentation, such as furfural and 5-hydroxymethylfurfural (HMF) (Delgenes et al., 1996; Modig et al., 2002). Lignin byproducts during biomass hydrolysis are also inhibitory to microorganisms. This mixture of inhibitors will likely be a great challenge to these fermenting strains. Many methods for detoxification have been investigated, including neutralization, overliming, evaporation, the use of ion-exchange resins, and activated charcoal adsorption (Ranatunga et al., 1997; Carvalho et al., 2004). Strain adaptation has also been successful by addition of furfural and ethanol into media and is as shown in a pending US patent (Lefebvre et al., 2007, 2008). Sugar Maple hot-water extract hemicellulosic hydrolyzate was concentrated and fractionated by a Nano-filtration membrane process
T. Liu et al. / Biotechnology Advances 28 (2010) 602–608
in our lab. In this study, E. coli FBR5 was challenged by hot-water wood extract hemicellulosic hydrolyzate. After repeated strain adaptation, a new improved strain: E. coli FBHW was obtained. Fermentation experiments indicated that FBHW was resistant to the toxicity of hydrolyzate in a fermentation medium of concentrated hydrolyzate, and that xylose was completely utilized by the strain to produce ethanol. FBHW was grown in concentrated hydrolyzate without any detoxification treatment and yielded 36.8 g/L ethanol. 2. Materials and methods 2.1. Processing of hemicellulose hydrolyzate from sugar maple Sugar maple wood chips were extracted by hot water at about 160 °C for 2 h. The hot-water extraction was carried out in a 65 ft3 digester with a wood to liquor ratio about 1:4. The wood extract was concentrated about 10-fold by a Nano-filtration membrane. The concentrated wood extract was hydrolyzed at the temperature of 135 °C for 25 min with 1% (wt.%, [H+] = 0.204 mol/L as catalyst) sulfuric acid added. Acid-insoluble lignin was centrifuged out (CEPA High speed centrifuge Z81G, cylinder speed 16,000 rpm, cylinder diameter 125 mm, New Brunswick Scientific, NJ 08818-4005, USA). Hydrolyzate was neutralized by Ca(OH)2 and NaOH, and then fractionated by Nano-filtration. Two mixtures of acids were also tested for hydrolysis of concentrated hot-water wood extract. In order to maintain [H+] = 0.204 mol/L as catalyst, a carefully prescribed mixture of three acids (0.33% H2SO4 + 0.43% HNO3 + 0.25% HCl, wt.%) was added into the concentrated wood extract and temperature kept at 135 °C for 10 min. Hydrolyzate was then neutralized by a mixture of Ca(OH)2 (for 0.33% H2SO4) and NaOH (for 0.43% HNO3 + 0.25% HCl). Another batch of concentrated hot-water wood extract was hydrolyzed by a two acid mixture (0.5% H2SO4 + 0.65% HNO3, wt.%) at a temperature of 135 °C for 10 min, and then neutralized by Ca(OH)2. 2.2. Microorganism and cell growth E. coli FBR5 was maintained at 4 °C in modified Luria-Bertani (LB Broth, Lennox, Fisher Scientific, Fair Lawn, New Jersey, USA) agar solid medium containing 10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl and 15 g/L agar, adjusted to a pH of 7.0, supplemented with 4 g/L xylose
603
(MP Biomedicals, Solon, Ohio, USA) and 20 mg/L tetracycline (MP Biomedicals, Solon, Ohio, USA). In order to prepare inocula, 2 loops of cells were transferred to 125 mL screw capped plastic flasks (NALGENE, Rochester, NY, USA) containing 50 mL sterile liquid medium. The liquid medium contained 10 g/L tryptone, 5 g/L yeast extract and 5 g/L NaCl, adjusted to a pH of 7.0, supplemented with 10 g/L xylose and 20 mg/L tetracycline. LB broth and xylose stock solution (400 g/L) were autoclaved (HIRAYAMA HICLAVETM HV-110, Amerex Instruments, Lafayette, CA, USA) separately at 121 °C for 15 min, followed by cooling to room temperature (approximately 45 °C for the LB agar solid medium) and then mixed. Filter-sterilized (0.22 µm sterile filter, NALGENE, Rochester, NY, USA) tetracycline stock solutions (20 mg/mL) were added to the above concentrations. The liquid culture was incubated at 35 °C for 12–14 h on a rotary shaker (GYROMAXTM 747R, Amerex Instruments, Lafayette, CA, USA). Shake speed was set at 160 rpm. After growth, 5 mL of culture were used to inoculate 100 mL of sterile fermentation medium. 2.3. Strain adaptation and fermentation in flask Original E. coli FBR5 was maintained at 4 °C on modified LB plate. In order to prepare active cells, a colony was transferred to 50 mL sterile liquid medium and incubated overnight at 35 °C. Strain adaptation was performed by exposing an FBR5 overnight culture to fermentation medium containing 10% (v/v) concentrated hydrolyzate and plating out dilutions on selective medium containing 10% (v/v) hydrolyzate. The selective medium contained the same ingredients as the solid medium but replaced xylose with concentrated hot-water wood extract hydrolyzate at different levels. The largest colonies were selected and plated on selective medium of the same concentrated hydrolyzate level in comparison with the parent. Inoculated Petri dishes were incubated at 35 °C (IncuMax IC150, Amerex Instruments, Lafayette, CA, USA). Colonies bigger than the parent were selected for fermentation test in a fermentation medium containing 20% (v/v) concentrated hydrolyzate. The strain of highest ethanol production was selected for dilution and plating on selective medium containing 20% (v/v) concentrated hydrolyzate. The selected strain was passed through concentrated hydrolyzate challenges several times while increasing hydrolyzate concentration gradually from 10% to 20%, 30%, 40% and 50%. The final strain obtained after successive adaptation, E. coli FBHW, was stored and used in the subsequent fermentation study.
Fig. 1. 600 MHz 1H-NMR spectrum for the concentrated hot-water wood extract hydrolyzate before membrane separation.
604
T. Liu et al. / Biotechnology Advances 28 (2010) 602–608
Fig. 2. 600 MHz 1H-NMR spectrum for the concentrated hot-water wood extract hydrolyzate after membrane separation.
Fermentations were performed in 125 mL screw capped plastic flasks containing 100 mL sterile fermentation medium at 35 °C for 96–144 h on a rotary shaker (shake speed set at 160 rpm). Samples were taken routinely until fermentation stopped. The fermentation medium contained the same ingredients as the liquid medium for inocula but replaced xylose with concentrated hot-water wood extract hydrolyzate at different levels. 2.4. Ion-exchange treatment of hydrolyzate C-100H, strong acid cation exchange resin, and A-400, strong base anion exchange resin (Purolite, Bala Cynwyd, PA, USA), were used for desaltification of dilute sulfuric acid wood extract hydrolyzate. The process of desaltification was carried out in 250 mL chromatographic column. After ion-exchange, hydrolyzate was adjusted to pH7.0 with Ca(OH)2 for fermentation study. 2.5. Fermentation in bioreactor Fermentations with pH control were carried out in a 1 L New Brunswick Bioreactor (BIOFLO 110; New Brunswick Scientific Co., New Brunswick, NJ, USA). Fermentation pH was controlled at 7.0 using 2.5 mol/L NaOH solution. The working volume of the bioreactor was 800 mL. LB Broth (16 g) (Lennox, Fisher Bioreagents, Fair Lawn, NJ, USA) was dissolved in X mL of distilled water in the bioreactor. The bioreactor was autoclaved at 121 °C for 15 min followed by cooling it to room temperature. (800 − X) mL of concentrated hydrolyzate was sterilized separately by autoclaving at 121 °C for 15 min in a glass bottle or passing through 0.22 µm sterile filter (nitrocellulose membrane, Millipore) which was held on a filter holder with receiver (1000 mL, NALGENE, Rochester, NY, USA). Different sugar concentrations of fermentation media were achieved by changing the values of X. For example, 200, 280 and 360 mL of distilled water for the media containing 136, 111 and 96.7 g/L reducing sugar, respectively. After cooling, concentrated hydrolyzate was added to the bioreactor and adjusted to pH 7.0 prior to inoculation. The bioreactor was inoculated with 40 mL of actively growing 12–14 h-old culture and incubated at 35 °C. Agitation speed was set at 200 rpm and air flow rate was set as 25 ccm.
2.6. Analytical methods Cell density (g/L) was estimated by using a predetermined correlation between dry weight cell concentrations (oven dry at 105 °C) versus optical density. An OD of one is equivalent to 0.31 g/L of dry cells (data not shown). One mL of fermentation broth was centrifuged to harvest cells. Cells were then washed twice with 1 mL 0.9% NaCl solution to get rid of color from the hydrolyzate. Optical density was measured at 540 nm after suspending the cells with 1 mL 0.9% NaCl solution. Cell numbers in the fermentation broth were counted under a microscope (Model: 12-575-251, Fisher Scientific, Fair Lawn, New Jersey, USA) (Shuler and Kargi, 2001). Ethanol concentration was measured by GC using Thermo Scientific Focus GC systems equipped with a Triplus automatic sampler and a TRACE TR-WaxMS (30 m × 0.25 mm × 0.25 µm) GC column. The GC oven was set to an initial temperature of 50 °C and held for 3 min, after which the temperature was increased at a rate of 30 °C per min until it reached a final temperature of 180 °C. The injector temperature was set as 200 °C and run on split mode with a constant flow of 1.5 mL/min. Samples were prepared by adding 100 µL of sample and 100 µL of internal standard (t-butanol 5.5 g/L) with 800 µL of distilled water. Total reducing sugars were quantified using 3,5-dinitrosalicylic acid method (DNS method, ACROS, New Jersey, USA) (Hu et al., 2008). Xylose concentration was measured by enzymatic assay kit (Megazyme, D-Xylose assay kit, Bray, Co. Wicklow, Ireland, 2008). For ethanol and sugar analysis, samples (1 mL) were taken intermittently and clarified by centrifugation (Micro-centrifuge 235C, Fisher
Table 1 Inhibitors chemical shift and concentration from 600 MHz 1H-NMR spectra. Inhibitor
Chemical shift ppm
Peak area
Concentration, g/L
Before
After
Before
After
Acetic acid Formic acid HMF Furfural
2.1 8.25 9.4 9.5
6651.45 N/A 7.30 111.31
371.06 N/A N/A N/A
38.86 N/A 0.27 3.12
2.17 N/A N/A N/A
T. Liu et al. / Biotechnology Advances 28 (2010) 602–608
Fig. 3. E. coli FBR5 in concentrated hot-water wood extract hydrolyzate medium containing 59.1 g/L reducing sugar.
Scientific, Fair Lawn, New Jersey, USA) at 13,600 × g for 5 min to remove cells. Supernatants were stored in eppendorf tubes at − 20 °C and analyzed at the end of each batch fermentation. Fermentation inhibitors (acetic acid, formic acid, furfural and HMF) concentrations in the concentrated wood extract hydrolyzate were determined by 600 MHz 1H-NMR (Bruker BioSpin Corporation, Billerica, MA, USA). After centrifugation, samples were prepared by adding 100 µL of sample and 100 µL of internal standard (TSP, SigmaAldrich, St Louis, MO, USA) with 800 µL of distilled water. Samples were homogenized in a mixer. The results were calculated by the ratio of inhibitors and TSP's peak area (Kiemle et al., 2004; Stipanovic and Kiemle, 2002; Copur et al., 2003). The NMR operating conditions are as follows, Probe Type: Broadband Observe Probe; Temperature: 30 °C; 90° Pulse: 11 μs; Interval between pulses: 10 s; Time for acquisition: 2.73 s; Sweep Width: 10 ppm; Center of spectrum: 4.5 ppm; Reference: Acetone at 2.2 ppm. The area of each peak was integrated by MestReNova software and the integral of TSP was set as 100. The concentration of total phenols in the concentrated wood extract hydrolyzate was measured by 4-aminoantipyrine colorimetric method (Standard Methods Committee, 1988). Ion concentrations of the concentrated wood extract hydrolyzate were analyzed by inductively coupled plasma (ICP). Samples were analyzed on a Perkin Elmer 3300DV Inductively Coupled Plasma Emission Spectrometer. Samples were diluted 100-fold with 2% nitric acid for analysis. The element concentrations were obtained via emissions from the following lines (in nanometers): Fe — 238.204; Ca — 317.933; Na — 589.952; S — 181.975; Cr — 205.960; Cu — 324.752; Mo — 202.031; Ni — 231.604; K — 766.490; P — 178.221; Mg — 285.213. Ethanol productivity was calculated as the maximum ethanol produced (g/L) divided by the fermentation time (h) to achieve maximum ethanol concentration and is expressed in g/(L ⁎ h). Ethanol yield (YP/S) was calculated as the maximum ethanol produced (g) divided by total reducing sugar consumed (g).
605
Fig. 4. Sugar utilization during fermentation of untreated and desalted hydrolyzate.
3. Results and discussion 3.1. 600 MHz 1H-NMR analysis for the concentrated hot-water wood extract hydrolyzate before and after membrane separation The acetyl groups from hemicellulose contribute to acetic acid formation in the extraction liquor. Our experimental results show that the pH of extraction liquor drops from the initial neutral conditions (∼pH7.0) to acidic conditions (∼pH3.2). This acidic condition during the hot-water extraction process further catalyzes the extraction and hydrolysis reactions. The extraction proceeds slowly initially and faster as pH decreases during extraction. Therefore, the hot-water extraction reactions are referred to as autocatalysis, and sometimes as autohydrolysis. A major portion of hemicellulose was extracted in the form of soluble oligomers and monomers in the extraction liquor (Liu, 2008). Upon membrane processing, water and other low molecular weight compounds, e.g. acetic acid, formic acid, methanol, furfural, etc., were preferentially sent to the permeate stream, while sugar monomers and oligomers along with high molecular weight compounds were concentrated in the concentrate stream. Xylose and acetic acid are the largest components recovered from the hydrolysis process.
Table 2 Metal ion, inhibitor and sugar concentration of the hydrolyzate before and after ionexchange.
Untreated Treated
Na, ppm
S, ppm
Ca, ppm
Xylose, g/L
Reducing sugar, g/L
Phenol, mg/L
8548 520.3
4460 318.9
414 4948
37.5 34.5
111.6 105.3
438.4 288.0
Fig. 5. Ethanol production during fermentation of untreated and desalted hydrolyzate.
606
T. Liu et al. / Biotechnology Advances 28 (2010) 602–608
Table 3 Ion concentration (ppm) of the hydrolyzate catalyzed by different mixed acids.
2-mixed acids 3-mixed acids
Na
S
Ca
Fe
6 275
902 1422
6106 3800
1819 3300
In 600 MHz 1H-NMR spectrum, the peaks of acetic acid, formic acid, HMF and furfural appeared at 2.1, 8.25, 9.4 and 9.5 ppm, respectively. As can be seen in Fig. 1, the peak area of acetic acid is very high (6651.45 at 2.1 ppm). The concentration of acetic acid is 38.86 g/ L. After membrane separation, a significant amount of the inhibitors have been removed from the hydrolyzate. No formic acid, furfural and HMF are detected by 600 MHz 1H-NMR (Fig. 2). Total phenol content decreased from 940.8 mg/L to 404.8 mg/L after membrane filtration. Reducing sugar concentration was 212 g/L. The data on chemical shift and concentration of inhibitors in the concentrated hot-water wood extract hydrolyzate before and after membrane separation are summarized in Table 1. 3.2. Fermentation of concentrated hot-water wood extract hydrolyzate catalyzed by dilute sulfuric acid
Fig. 7. Fermentation results of concentrated (3-mixed-acid) hydrolyzate medium containing 134 g/L reducing sugar.
comparison with results shown in Fig. 3, this one shows half of the productivity and yield found for hydrolysate medium containing 59.1 g/L reducing sugar.
After repeated strain adaptation, FBR5's growth was slightly inhibited in the concentrated hydrolyzate medium containing 59.1 g/L total reducing sugar (15.2 g/L xylose, data not shown in the figure). In Fig. 3, one can observe that cells grew slowly in the first two days, and then reached the exponential growth phase in the third day, at which point xylose utilization rate decreased. During the first day, 0.2 g/L of xylose were utilized. It took 4 days to consume all the xylose and 29.2 g/L reducing sugar in the medium. Only 0.47 g/L ethanol was produced in the first day of fermentation. Ethanol concentration increased over the next few days, and a maximum concentration of 9.30 g/L was achieved on the fifth day. Ethanol productivity was 0.078 g/(L ⁎ h) with a yield (YP/S) of 0.32 g ethanol/g consumed reducing sugar. After strain adaptation, a mutant was isolated and named as E. coli FBHW. FBHW was used in subsequent fermentation studies. In further research, FBHW's growth and ethanol production were found to be significantly inhibited in more concentrated hot-water wood extract hydrolyzate medium containing 111.6 g/L reducing sugar (containing 37.5 g/L xylose). During fermentation over four days, 13.73 g/L of xylose and 23.3 g/L of reducing sugar was utilized and 3.49 g/L of ethanol was produced. Ethanol productivity was 0.036 g/(L ⁎ h) with a yield (YP/S) of 0.15 g ethanol/g consumed reducing sugar. In
According to the 600 MHz 1H-NMR result most of the fermentation inhibitors have been removed from the hydrolyzate, yet cell growth was found to be still inhibited. Na2SO4 was formed during neutralization of hydrolyzate, and it cannot pass through the nanofiltration membrane. To investigate inhibition effect of Na2SO4 on fermentation, the hydrolyzate was treated by an ion-exchange resin, then adjusted pH to 7.0 with Ca(OH)2 for fermentation studies. One can see from Table 2 that the concentrations of Na and S decreased after ion exchange treatment. The concentration of Ca increased, due to adjustment of pH with Ca(OH)2. Concentrations of xylose and reducing sugar all decreased slightly, from 37.5 and 111.6 g/L to 34.5 and 105.3 g/L, respectively. The loss rate of xylose and reducing sugar is 8% and 5.6%, respectively. Total phenol content also dropped from 438.4 to 288.0 mg/L. In Fig. 4 we can see that xylose and reducing sugar was utilized quickly and thoroughly by E. coli after ion-exchange treatment. In the fermentation of treated hydrolyzate 34.3 g/L of xylose was consumed after 72 h of fermentation, with 50.8 g/L of reducing sugar being
Fig. 6. Fermentation results of concentrated (2-mixed-acid) hydrolyzate medium containing 118 g/L reducing sugar.
Fig. 8. Fermentation results of concentrated (3-mixed-acid) hydrolyzate medium containing 111 g/L reducing sugar.
3.3. Fermentation of dilute sulfuric acid hydrolyzate after ion-exchange
T. Liu et al. / Biotechnology Advances 28 (2010) 602–608
Fig. 9. Fermentation results of autoclaved concentrated (3-mixed-acid) hydrolyzate media containing 136 or 96.7 g/L reducing sugar.
consumed. In the fermentation of hydrolyzate without ion-exchange treatment 13.7 g/L xylose was consumed after 96 h of fermentation, with 23.3 g/L of reducing sugar being consumed. From Fig. 5 we can see that the highest ethanol concentration was 22.6 g/L after 96 h (4 days) fermentation of the treated hydrolyzate. The ethanol productivity was 0.24 g/(L ⁎ h) with a yield (YP/S) of 0.45 g ethanol/g consumed reducing sugar. In the fermentation of untreated hydrolyzate 3.49 g/L ethanol was produced after 96 h of fermentation. The ethanol productivity for untreated hydrolyzate was 0.036 g/(L ⁎ h) with a yield (YP/S) of 0.15 g ethanol/g consumed reducing sugar. 3.4. Fermentation of hydrolyzate catalyzed by dilute mixed acids Table 3 shows the concentration of Na, S, Ca and Fe. Fig. 6 shows that the highest ethanol concentration of 2.62 g/L was achieved at the second day of fermentation, and then decreased during the next 2 days of fermentation. The high concentration of Ca may be a cause of cell growth and ethanol production inhibition. Only 2.34 g/L of ethanol was produced and only 15 g/L of reducing sugar was consumed after 96 h of fermentation. The ethanol productivity was 0.055 g/(L⋅h) with
607
a yield (YP/S) of 0.26 g ethanol/g consumed reducing sugar after 48 h of fermentation. Strain adaptation is continuing. The last results reported here are shown in Fig. 7. The hydrolyzate produced from a 3-mixed acid hydrolysis was concentrated to 134 g/L of reducing sugar by nanofiltration membrane and used as the fermentation substrate, directly without any detoxification pretreatment. As can be seen in Fig. 7, sugar concentration decreased gradually, and 108 g/L reducing sugar was utilized after 96 h of fermentation. The maximum ethanol concentration of 36.9 g/L was achieved at the end of 96 h of fermentation. Ethanol productivity was 0.38 g/(L ⁎ h) with a yield (YP/S) of 0.34 g ethanol/g consumed reducing sugar. The extract hydrolyzate at 134 g/L reducing was diluted with distilled water to 111 g/L reducing sugar and the fermentation was tested again. Higher ethanol productivity was achieved by using this less concentrated hydrolyzate as substrate (Fig. 8), most likely due to lower concentrations of sugar and inhibitors. When the reducing sugar content of the hydrolyzate medium was adjusted to 111 g/L, 34.8 g/L of ethanol was produced and 90.1 g/L of reducing sugar was consumed during 72 h of fermentation. The ethanol productivity was 0.48 g/(L ⁎ h) with a yield (YP/S) of 0.39 g ethanol/g consumed reducing sugar. 3.5. Comparison of sterilization methods for hydrolyzate The effect of different hydrolyzate sterilization methods on cell growth and ethanol production was investigated. From Fig. 9 we can see that E. coli utilized sugar and produced ethanol slowly in media containing hydrolyzate that had been autoclaved. In the fermentation of hydrolyzate medium containing 136 g/L reducing sugar, 73 g/L reducing sugar was consumed after 144 h of fermentation. Ethanol concentration increased slowly, with only 0.27 g/L ethanol produced after 48 h of fermentation. The maximum ethanol concentration of 20.1 g/L was achieved after 96 h of fermentation and decreased thereafter. The ethanol productivity was 0.21 g/(L ⁎ h) with a yield (YP/S) of 0.34 g ethanol/g consumed reducing sugar. In the fermentation of hydrolyzate medium containing 96.7 g/L reducing sugar, the maximum ethanol concentration of 32.0 g/L was achieved after 96 h of fermentation, with 78.5 g/L reducing sugar being consumed. The ethanol productivity was 0.33 g/(L ⁎ h) with a yield (YP/S) of 0.41 g ethanol/g consumed reducing sugar. Fig. 10, in comparison with Fig. 2,
Fig. 10. 600 MHz 1H-NMR spectrum for the concentrated hot-water wood extract (3-mixed-acid) hydrolyzate after autoclaving.
608
T. Liu et al. / Biotechnology Advances 28 (2010) 602–608
shows that acetic acid concentration increased while polymer acetyl group decreased upon autoclaving. A small amount of furfural and sediment was formed during hydrolyzate autoclaving. The concentration of acetic acid and furfural was 2.63 and 0.25 g/L, respectively, in the autoclaved hydrolyzate containing 212 g/L reducing sugar. 4. Concluding remarks In conclusion, E. coli FBR5 can be adapted to hot-water wood extract hydrolyzate (containing 59.1 g/L reducing sugar) catalyzed by dilute sulfuric acid and can utilize xylose completely after strain adaptation; however, E. coli's growth is still strongly inhibited in more concentrated hydrolyzate containing 111.6 g/L reducing sugar. Inhibition is decreased after ion-exchange. In the fermentation of hydrolyzate catalyzed by 3-mixed-acids, cells grow fast and produce 36.9 g/L ethanol after 96 h of culture. The hydrolyzate catalyzed by 2-mixed-acids shows significant inhibition on cell growth and ethanol production. The concentration of acetic acid and furfural is increased during hydrolyzate autoclaving. The autoclaved hydrolyzate exhibits some increased inhibition of cell growth and ethanol production. Acknowledgements The authors are indebted to Dr. Bruce Dien for providing the Escherichia coli FBR5 samples to carry out this study. USDOE, NYSERDA and China National Key Research Program (2010CB732201) are gratefully acknowledged for their financial supports. The authors are grateful for the staff in the Bioprocess Engineering lab, SUNY ESF for their support in completing this research. Dr. Thomas E. Amidon is especially acknowledged for his support in getting this work done. We would also like to thank Mr. Dave Kiemle, Mr. Christopher D. Wood and Ms. Debra A. Driscoll for their help with these studies. References Beall DS, Ohta K, Ingram LO. Parametric studies of ethanol production from xylose and other sugars by recombinant Escherichia coli. Biotechnol Bioeng 1991;38:296–303. Carvalho Wde, Canilha L, Mussatto SI, Dragone G, Morales MLV, Solenzal AIN. Detoxification of sugarcane bagasse hemicellulosic hydrolysate with ion-exchange
resins for xylitol production by calcium alginate-entrapped cells. J Chem Technol Biotechnol 2004;79:863–8. Copur Y, Kiemle DJ, Stipanovic A, Koskinen J, Makkonen H. 1H-NMR spectroscopic determination of carbohydrates and yield in pine and maple pulps. Paperi ja Puu 2003;85:158–62. Delgenes JP, Moletta R, Navarro JM. Effects of lignocellulose degradation products on ethanol fermentations of glucose and xylose by Saccharomyces cerevisae, Zymomonas mobilis, Pichia stipitis, and Candida shehatae. Enzyme Microb Technol 1996;19:220–5. Dien BS, Nichols NN, O'Bryan PJ, Bothast RJ. Development of new ethanologenic Escherichia coli strains for fermentation of lignocellulosic biomass. Appl Biochem Biotechnol 2000;84:181–96. Dien BS, Cotta MA, Jeffries TW. Bacteria engineered for fuel ethanol production: current status. Appl Microbiol Biotechnol 2003;63:258–66. Hu RF, Lin L, Liu TJ, Liu SJ. Reducing sugar content in hemicellulose hydrolysate by DNS method: a revisit. J Biobased Mater Bioeng 2008;2(2):156–61. Ingram LO, Conway T, Clark DP, Sewell GW, Preston JF. Genetic engineering of ethanol production in Escherichia coli. Appl Environ Microbiol 1987;53(10):2420–5. Kiemle DJ, Stipanovic AJ, Mayo KE. Proton NMR methods in the compositional characterization of polysaccharides. ACS Symp series 2004;864:122–39. Lee RL, Paul JW, Van Z. Willem H,, Isak SP, Microbial Cellulose Utilization: Fundamentals Biotechnology. Microbiol Molec Biology Rev 2002;66(3):506–77. Lefebvre BG, Savelski MJ, Hecht GB. Ethanologenic bacteria, International application number: PCT/US2007/021892, 12 October 2007. Lefebvre BG, Savelski MJ, Hecht GB. Ethanol resistant and furfural resistant strains of E. coli FBR5 for production of ethanol from cellulosic biomass, International publication number: WO 2008/048513 A2, 24 April 2008. Liu SJ. A kinetic model on autocatalytic reactions in woody biomass hydrolysis. J Biobased Mater Bioeng 2008;2(2):135–47. Nan Fu, Paul P. Co-fermentation of a mixture of glucose and xylose to ethanol by Zymomonas mobilis and Pachysolen tannophilus. World J Microbiol Biotechnol 2008;24:1091–7. Martin Gregory JO, Knepper A, Zhou B, Pamment NB. Performance and stability of ethanologenic Escherichia coli strain FBR5 during continuous culture on xylose and glucose. J Ind Microbiol Biotechnol 2006;33:834–44. Megazyme, Bray, Co. Wicklow, Ireland, 2008; Website: http://secure.megazyme.com. Modig T, Liden G, Taherzadeh MJ. Inhibition effects of furfural on alcohol dehydrogenase, aldehyde dehydrogenase, and pyruvate dehydrogenase. Biochem J 2002;363:769–76. Qureshi N, Dien BS, Nichols NN, Saha BC, Cotta MA. Genetically engineered Escherichia coli for ethanol production from xylose: substrate and product inhibition and kinetic parameters. Inst Chem Eng: Food Bioproducts Process 2006;84(C2):114–22. Ranatunga TD, Jervis J, Helm RF, McMillan JD, Hatzis C. Toxicity of hard-wood extractives toward Saccharomyces cerevisiae glucose fermentation. Biotechnol Lett 1997;19:1125–7. Shuler ML, Kargi F. Bioprocess Engineering: Basic Concepts. Second Edition. NJ, USA: Prentice Hall; 2001. p. 156. Standard Methods Committee. 5530 Phenols. Aggregate organic constituents; 1988. 5000:5/48. Stipanovic A, Kiemle DJ. Proton NMR methods in the compositional characterization of hemicelluloses. Abstracts of Papers, 223rd ACS National Meeting; 2002. April 7–11.