Production of Butanol from Switchgrass using a Novel Detoxification Process

Transcription

1 Production of Butanol from Switchgrass using a Novel Detoxification Process Niblack Research Scholar B.S. in Biosystems Engineering, Oklahoma State University Graduation Date: May 09, 2015 [email protected] Research Advisor: Dr. Hasan K. Atiyeh Department of Biosystems and Agricultural Engineering, Oklahoma State University [email protected] Student Contestant: Date: 3/22/2015 Student Branch Advisor Date: 3/20/2015

2 Origin of Research The funding for this research was made available through the Niblack Research Scholars Program at Oklahoma State University. This prestigious scholarship is awarded to twelve undergraduate students annually. During the twelve month program I was able to conduct all of the pretreatment, hydrolysis, and fermentation necessary for my project under the supervision of Kan Liu, a Ph.D. candidate under Dr. Hasan Atiyeh. The results of this research are presented in this paper. The results of this experiment have also been presented at the American Society of Engineering Educators (ASEE) Midwest Region Rally, where it received the honor of Best Undergraduate Poster. This research has also been presented to the Vice President of Research at Oklahoma State University in order to complete the requirements of the Niblack Research Scholars program. I would like to sincerely thank Dr. Hasan Aityeh for serving as my faculty mentor. I would also like to thank Dr. Kan Liu and Mr. Oscar Pardo-Planas for serving as graduate mentors and assisting me with lab technique. I am extremely grateful for their support, encouragement, and guidance throughout the duration of this research project. I would also like to thank Dr. John and Heidi Niblack for their generous donation to the Niblack Research Program, which has allowed numerous students to participate in undergraduate research. I would like to further thank the Oklahoma Agricultural Experimental Station (OAES) and Sun Grant Initiative (US Department of Transportation) for additional funding. 2

3 Abstract The conversion of biomass to alcohols and organic acids is a rapidly developing sector of the biofuels industry. The process starts with pretreatment of biomass to allow access to cellulose and hemicellulose by cellulases. The enzymes hydrolyze cellulose and hemicellulose to C6 and C5 sugars. Through fermentation, C6 and C5 sugars from hydrolyzed biomass are converted into acetone, butanol and ethanol (ABE), as well as acetic acid, and butyric acid. The main product of the fermentation is butanol, which can be upgraded to jet fuels using chemical catalysts. In our earlier studies, direct conversion of pretreated switchgrass hydrolyzate to ABE was not possible due to presence of inhibitors in the hydrolyzate. An adsorbent material was kept in the medium during the batch fermentations using Clostridium acetobutylicum ATCC 824. The results showed that detoxified hydrolyzate consumed glucose at a similar level to pure sugar medium used as control, although not all the butanol could be detected due to adsorption. This shows the potential use of switchgrass for production of butanol for further processing into jet fuels. Keywords: Butanol production, switchgrass, enzymatic hydrolysis, fermentation, biofuels 3

4 Background Production of fuels for transportation from renewable resources without major changes to existing fuel infrastructure is necessary to reduce the cost of transition from fossil fuels to sustainable fuels. Butanol can easily be incorporated into the current gasoline infrastructure and has 25% more energy than ethanol, making it a more attractive biofuel 1. Butanol can additionally be upgraded to jet fuel using chemical catalysts 2. The production of butanol from lignocellulosic materials is considered a renewable source of energy. In the Acetone-Butanol-Ethanol (ABE) fermentations, clostridia microorganisms are used to ferment the sugars obtained from biomass feedstocks. Different feedstocks have been investigated as substrate for ABE fermentation, such as barley straw, corn stover, or wood residues 3. Among the potential biomass sources, switchgrass has been identified as a model herbaceous energy crop for the United States 4. It is a perennial grass native to North America and produces high biomass yields while requiring less water and fertilizer than other cool season grasses 5. The bioconversion of switchgrass to butanol is a promising technology but has been performed only with limited success 6, probably due to the presence of inhibitors from pretreatment and hydrolysis. Objective The objective of this project was to investigate the conversion of switchgrass into butanol, which can be upgraded to jet-fuel through hydrogenation. In order for this conversion to occur, switchgrass should be pretreated to access to cellulose and hemicellulose. This is followed by hydrolysis of cellulose and hemicellulose to C6 and C5 sugars. Switchgrass hydrolyzate should support the growth of Clostridium acetobutylicum ATCC 824 either with or without detoxification. The development of an effective separation process is required to separate butanol from other products after fermentation. 4

5 Method In order to meet the objectives, three sets of experiments were required. The first set of experiments was to pretreat switchgrass into sugars that can be fermented into butanol. The second set of experiments was to ferment the non-detoxified and detoxified switchgrass hydrolyzates. The final set of experiments was the recovery of butanol from the fermentation medium. The biomass used for this project was Alamo switchgrass (Panicum virgatum L.) harvested at the end of July of 2012 in Oklahoma. 2 mm particles were obtained by passing the switchgrass through a Thomas-Wiley mill (Arthur H. Thomas Co., Philadelphia, PA). 1. Pretreatment, Hydrolysis, and Compositional Analysis In order to prepare the switchgrass for fermentation it is necessary to first pretreat it to reduce cellulose crystallinity and association to lignin 7. This allows the enzymes used in hydrolysis to convert the sugar polymers into monomeric sugars such as glucose and xylose. A compositional analysis must be performed to determine the conversion effectiveness of pretreatment and to compare it with the theoretical potential of conversion during hydrolysis. Pretreatment of raw switchgrass was performed in a 1-L Parr reactor at 200 C for 10 minutes. After pretreatment, the pretreated switchgrass was washed four times; using 500 ml of warm DI water for each wash. For compositional analysis, the NREL protocol Determination of Structural Carbohydrates and Lignin in Biomass was used 8. The polymeric sugars and lignin compositions were calculated. This data allowed the calculation of the glucan-to-glucose conversion during hydrolysis. Enzymatic hydrolysis was performed for 48 hours using Accelerase 1500 at a loading of 50 FPU/g glucan, 50 C and 250 RPM. It 5

6 was conducted in 250 ml baffled flasks with a total working mass of 100 g with 14% (w/w) solid loading. Periodical samples were taken and analyzed using a high performance liquid chromatograph (HPLC) for sugar composition. After 48 hours, the hydrolyzate was frozen until used in fermentation experiments. 2. Fermentation of switchgrass hydrolyzate The ability of C. acetobutylicum ATCC 824 to produce butanol from switchgrass hydrolyzates was investigated by a fermentation experiment with four treatments. Treatment 1 used non-detoxified switchgrass hydrolyzate. Treatment 2 was switchgrass hydrolyzate with the adsorbent in the fermentation bottle to remove inhibitors, i.e., soak up the inhibitors in the medium. Each one of these treatments had a paired control treatment that contained glucose as sugar source instead of hydrolyzate. Control 1 contained pure glucose, and control 2 contained glucose with the adsorbent in the fermentation bottles. C. acetobutylicum ATCC 824 spores were heat shocked at 75 C for 10 min and cooled on ice for 2 min, and then the microorganism was passaged twice on a lean sugar medium for activation. Fermentations were performed in an anaerobic chamber at 35 C. Samples were taken every 12 hours for the 72 hours of fermentation. Solvent analysis was performed using a gas chromatography (GC). Sugar analysis was done using HPLC. Cell growth was followed using a spectrometer at a wavelength of 600 nm. Each treatment was performed in triplicate to ensure consistency and accuracy in the data. 3. Separation of butanol from fermentation medium After fermentation is completed butanol should be recovered from the fermentation medium. This was achieved using a Bucchi Rotovapor system. The Rotovapor uses a vacuum pump 6

7 in order to lower the pressure inside the evaporating flask. When the vapor pressure of a solvent is reached, it is extracted from the evaporating flask and condensed on a condenser column, which drips into the collecting flask. Numerous conditions were tested in the Rotovapor in order to optimize the extraction of butanol from the fermentation medium. In order to calculate the recovery percentages of butanol, the following equations were used: %Butanol (BuOH) evaporated = (C BuOH,i V soln,i ) (C BuOH,F V soln,f ) (C BuOH,i V soln,i ) %Butanol Recovered in Aqueous (Aq) Phase = (C BuOH.Aq V BuOH,Aq ) (C BuOH,i V soln,i ) % Butanol Recovered in Organic (org)phase = (C BuOH,Org V BuOH,Org ) (C BuOH,i V soln,i ) Where V is volume (ml) and C is concentration (g/l), subscript F is final, subscript i is initial. Results 1. Pre-treatment, Hydrolysis, and Compositional Analysis After pretreatments were finished compositional analysis was performed to measure the percentages of sugar polymers, as well the lignin content. Table 1 shows the composition of raw and pretreated switchgrass. The glucan content increased from 35% before pretreatment to 56% after pretreatment. A mass balance indicated that only 16% of the glucan initially present was lost during pretreatment, and 93% of the xylan was removed. The glucan to glucose conversion during hydrolysis can be seen in Figure 1. After 48 hours, almost 92% of the available glucan was converted to glucose or other sugars. 7

8 Glucan-to-glucose Conversion (%) KK Barnes Student Paper Competition Table 1- The compositions of raw and pretreated switchgrass 9 Compound Composition switchgrass before pretreatment (%db a ) Composition of pretreated switchgrass (%db a ) Glucan ± ± 0.59 Xylan ± ± 0.09 Galactan 1.34 ± ± 0.07 Arabinan+Mannan 2.41 ± ± 0.01 Lignin ± ± 0.27 Extractives 6.93 ± 0.04 ND b a db: dry basis b ND: Not determined Time (h) Figure 1- Percent glucan conversion with respect to time 9 2. Fermentation of switchgrass hydrolyzate Figure 2 shows the cell growth over 72 hours of fermentation. As it can be seen in Figure 2, the hydrolyzate with the adsorbent showed the best growth. This suggests that the adsorbent 8

9 removed inhibitors, allowing more rapid growth of cells than in the non-detoxified switchgrass hydrolyzate. However, this increase in growth did not reflect in higher butanol concentrations as shown in Figure 3. This disparity is possibly caused by the adsorbent binding butanol and other solvents. Previous tests indicated that the adsorbent was capable of adsorbing around 80% of butanol present in the solution (data not shown). Accounting for the butanol adsorbed, a concentration of approximately 15 g/l could have been reached in the fermentation medium. This result is consistent with the pure sugar control. Figure 4 shows the total acetone, butanol and ethanol (ABE) concentrations produced. Once again, the treatments with adsorbent in the bottle showed a lower observed ABE concentration. Figure 5 shows the sugar consumption in each treatment. C. acetobutylicum grown on hydrolyzate with adsorbent consumed all sugars present in 48 hours. This is a 25% higher substrate consumption compared to the treatments with no adsorbent. Figure 6 shows the total acid concentrations throughout the fermentation. Table 2 shows the butanol and ABE yields and productivities for all treatments. For treatments with adsorbent, projected butanol productivity was estimated assuming that 80% of butanol could have been adsorbed Pure Sugar Non-Detoxified Hydrolysate Pure Sugar & Adsorbent Hydrolysate & Adsorbent OD Time (h) Figure 2- Optical density (OD 600 ) of broth during fermentation. 9

10 Concentration (g/l) Concentration (g/l) KK Barnes Student Paper Competition Pure Sugar Non-Detoxified Hydrolysate Pure Sugar & Adsorbent Hydrolysate & Adsorbent Time (h) Figure 3- Butanol concentration for each treatment during the fermentation Pure Sugar Non-Detoxified Hydrolysate Pure Sugar & Adsorbent Hydrolysate & Adsorbent Time (h) Figure 4- Total acetone, butanol and ethanol (ABE) concentrations for each treatment during fermentation. 10

11 Concentration (g/l) Concentration (g/l) KK Barnes Student Paper Competition Pure Sugar Pure Sugar & Adsorbent Non-Detoxified Hydrolysate Hydrolysate & Adsorbent Time (h) Figure 5-Total sugar (Glucose, Xylose, Mannose, and Arabinose) concentrations during the fermentation Pure Sugar Pure Sugar & Adsorbent Non-Detoxified Hydrolysate Hydrolysate & Adsorbent Time (h) Figure 6 - Total acid concentration during the fermentation. 11

12 Table 2- Butanol productivity of the fermentation after 72 hours of fermentation. ABE Produced (g/l) ABE Productivity (g/l h) Treatment Butanol Productivity (g/l h) ABE Yield (g/g) Pure Sugar Pure Sugar & Adsorbent 0.20 a a 0.32 a 0.25 a Non-Detoxified Hydrolyzate Hydrolyzate & Adsorbent 0.22 a a 0.34 a 0.29 a a Calculations were made assuming 80% of butanol was adsorbed 3. Separation of butanol from fermentation medium In order to develop an appropriate separation technique certain conditions for the rotary evaporator operation were studied. Preliminary studies were conducted to determine the pressure at which butanol began to boil from solutions. In addition, further tests showed that 150 RPM was effective at removing butanol from solution. With these favorable operating conditions for the extraction of butanol, a simulated fermentation medium was prepared containing 13 g/l butanol, 5 g/l acetone, 2 g/l butyric acid, and 2 g/l acetic acid. The simulated fermentation medium was then placed in the Rotovapor and the butanol extraction program was executed. Table 3 shows the mass recovery of butanol from the simulated fermentation medium. It can be seen that 62.2% of butanol was evaporated, and 58.8% was recovered. This shows that the evaporation system was working effectively. The collected aqueous fraction was mostly butanol, water, and acetone. However, the collected organic fraction was mainly butanol. Due to the low boiling point of acetone, it is easily separated from butanol and water using either traditional distillation or Rotovapor separation. 12

13 Table 3- Solvent recovery data for Rotovapor extraction of the simulated fermentation medium. Initial Butanol Conc. (g/l) Evapor ated (g) Collec ted (g) Evapor ated (ml) Collected Org/Total (ml/ml) % Butanol Evaporated (w/w) % Butanol Recovered Aqueous (w/w) % Butanol Recovered Organic (w/w) Total %Butanol Recovered (w/w) Simulated Fermentation Medium / Conclusions Butanol was successfully produced to a concentration of 6.5 g/l in the non-detoxified switchgrass hydrolysate. The addition of an adsorbent increased the sugar consumption of Clostridium acetobutylicum ATCC 824 by 25%. The addition of an adsorbent also reduced the butanol concentration dissolved in the fermentation medium, which can potentially favor increased butanol production and warrants further investigation. Publications and Presentations The results from this Niblack project have been presented in the poster form at 2013 OSU BMBGSA Colloquium, 2014 OSU Research Week, 2014 Oklahoma ASABE Sectional Meeting, 2014 ASABE Annual International Meeting (Montreal, QC), and 2014 ASEE Midwest Region Conference (Fort Smith, Arkansas). 13

14 References 1. Jin, C.; Yao, M.; Liu, H.; Lee, C.-f. F.; Ji, J. (2011) Progress in the production and application of n-butanol as a biofuel. Renewable and Sustainable Energy Reviews, 15 (8), Harvey, B. G.; Meylemans, H. A.(2011) The role of butanol in the development of sustainable fuel technologies. Journal of Chemical Technology and Biotechnology, 86 (1), Qureshi, N.; Saha, B. C.; Dien, B.; Hector, R. E.; Cotta, M. A. (2010) Production of butanol (a biofuel) from agricultural residues: Part I Use of barley straw hydrolysate. Biomass and bioenergy, 34 (4), Sanderson, M. A.; Adler, P. R.; Boateng, A. A.; Casler, M. D.; Sarath, G. (2006) Switchgrass as a biofuels feedstock in the USA. Canadian Journal of Plant Science, 86 (Special Issue), Samson, R. A.; Omielan, J. A. (1992) In Switchgrass: A potential biomass energy crop for ethanol production, The Thirteenth North American Prairie conference, Windsor, Ontario, pp Qureshi, N.; Saha, B. C.; Hector, R. E.; Dien, B.; Hughes, S.; Liu, S.; Iten, L.; Bowman, M. J.; Sarath, G.; Cotta, M. A. (2010) Production of butanol (a biofuel) from agricultural residues: Part II Use of corn stover and switchgrass hydrolysates. biomass and bioenergy 34 (4), Wyman, C. E.; Dale, B. E.; Elander, R. T.; Holtzapple, M.; Ladisch, M. R.; Lee, Y. (2005) Coordinated development of leading biomass pretreatment technologies. Bioresource technology 96 (18), Sluiter, A.; Hames, B.; Ruiz, R.; Scarlata, C.; Sluiter, J.; Templeton, D.; Crocker, D. (2008) Determination of structural carbohydrates and lignin in biomass. Laboratory analytical procedure. 9. Liu, K., Atiyeh, H. K., Pardo-Planas, O., Ezeji, T., Ujor, V., Overton, J.C., Berning, K., Wilkins, M.R., Tanner, R.S. (2015) Butanol Production from Hydrothermolysis-Pretreated Switchgrass: Quantification of Inhibitors and Detoxification of Hydrolyzate. Bioresource Technology Technology. 14