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Conditioning hemicellulose hydrolysates for fermentation: Effects of overliming pH on sugar and ethanol yields
Process Biochemistry 41 (2006) 1806–1811 www.elsevier.com/locate/procbio
Conditioning hemicellulose hydrolysates for fermentation: Effects of overliming pH on sugar and ethanol yields Ali Mohagheghi *, Mark Ruth, Daniel J. Schell National Renewable Energy Laboratory, National Bioenergy Center, 1617 Cole Blvd., Golden, CO 80401, USA Received 19 January 2006; received in revised form 21 March 2006; accepted 24 March 2006
Abstract Overliming is an effective way of conditioning to reduce the toxicity of hydrolysates generated from pretreatment of lignicellulosic biomass for ethanol production. In this work, a range of target overliming pH values from 9 to 11 was studied, and xylose fermenting Zymomonas mobilis strain 8b was used to evaluate the fermentability of overlimed corn stover hemicellulose hydrolysate. pH 11 overlimed hydrolysate was highly fermentable, but xylose losses were the greatest at this condition. Based on ethanol yield and fermentative xylose conversion, pH 10-conditioned hydrolysate produced the best results, 75% xylose utilization and 76% ethanol yield. This condition also produces the highest overall ethanol yield based on total sugars available in the unconditioned hydrolysate, 70%. Overall mass balance closures were very good averaging between 97 and 100% for all experiments. Calcium and sulfur mass balance closures ranged from 75 to 90% and indicated that approximately 50% of the calcium ends up in the gypsum cake, which is calculated to be approximately 63% gypsum. Overall result shows that the pH of the overliming process is the key factor for improving hydrolysate fermentability but too high a pH destroys some of the available sugars and reduces overall ethanol yield. Thus, it is critical to keep the overliming pH as low as possible while making the hydrolysate fermentable. # 2006 Elsevier Ltd. All rights reserved. Keywords: Lime; Gypsum; Conditioning; Corn stover; Zymomonas mobilis; Mass balance
1. Introduction In the context of enzymatically converting lignocellulosic biomass to ethanol utilizing a dilute acid pretreatment step to produce xylose-rich liquor and to enhance cellulose digestibility, a conditioning step is typically required to improve hydrolysate fermentability [1,2]. Many conditioning processes involving chemical, physical and biological methods have been proposed to reduce hydrolysate toxicity prior to fermentation [2–4]. These processes include pH adjustment with lime (calcium oxide) and other caustics [4–10], removal of volatiles using steam stripping and other processes [11], ion exchange [7,12], extraction [12], and adsorption [7,13]. The process most widely used for hydrolysate conditioning is treatment with lime [9,14–17], a process hereafter referred to as ‘‘overliming’’. A study by Larsson (1999) [2] found that overliming is the most cost effective method for detoxifying soft wood hydrolysates. The overliming process begins by
* Corresponding author. Tel.: +1 303 384 6175; fax: +1 303 384 6877. E-mail address: [email protected] (A. Mohagheghi). 1359-5113/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2006.03.028
adding lime to adjust the pH of the hydrolysate liquor to a high value, typically in the range of 9–11. The liquor is usually heated to a temperature in the range of 50–60 8C. Even though lime hydration is a highly exothermic reaction, heating may still be required to reach the desired temperature. Once the pH and temperature are at the target values, the solution is held at temperature for a period of time (typically around 30 min), then filtered to remove the precipitate gypsum (calcium sulfate) formed by divalent calcium from the lime combining with sulfate in the hydrolysate. Finally, re-acidifying the hydrolysate to a value appropriate for fermentation completes the process. The mechanism by which overliming reduces hydrolysate toxicity is not well understood. Agblevor et al. [18] used 13C NMR to determine that overliming removes aliphatic and aromatic acid and other aromatic and aliphatic compounds. The exothermicity of lime hydration and the heating of the hydrolysate during the process decrease the solubility of gypsum and helps to strip off volatile compounds such as furfural [4,19]. A potential drawback of overliming is sugar degradation due to hydroxide-catalyzed degradation reactions; however, overliming to a higher pH produces less toxic (more fermentable) liquor [20].
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A conceptual design was developed at the National Renewable Energy Laboratory for converting corn stover to ethanol using dilute acid prehydrolysis, enzymatic cellulose saccharification, and co-fermentation of biomass sugars [21,22]. Material and energy balances were modeled assuming a 30% solids loading in pretreatment and 20% initial total solids loading into enzymatic saccharification. Overliming conditions and effects were not well understood when the process model was developed but it was assumed that in the overliming step, all of the sulfuric acid in the hydrolysate reacted with lime to produce gypsum. All gypsum was assumed to be insoluble and removed by solid/liquid separation prior to enzymatic hydrolysis and fermentation. The reactions between the acid and caustic streams and the hydrolysate liquor did not consider potential sugar losses during overliming. Also, process model did not take into account that any soluble gypsum could affect other downstream unit operations (e.g., distillation preheaters). The purpose of this study was three-fold. First, to determine how conditioning pH over a range of values from 9 to 11 impacts sugar degradation. Second, to determine the overliming pH that enables the highest ethanol yield. Third, to perform overall mass and calcium and sulfur elemental balances to quantify the partitioning of these elements during the overliming process. While we fixed the overliming temperature and hold time and thus did not attempt to optimize the conditioning step, we sought to better understand the likely features and tradeoffs encountered in a potential industrial process.
2. Materials and methods 2.1. Hydrolysate The hemicellulose hydrolysate used in this work was produced from corn stover using a dilute sulfuric acid pretreatment process carried out in a 1 tonnes/ d pilot-scale continuous reactor operating at a solids concentration of 25% (w/ w), temperature of 190 8C, and an approximate residence time of 1 min, as reported previously [23]. Pretreatment liquor was produced by centrifuging (Sorvall Evolution, Kendro, Asheville, NC) the slurry at 8500 rpm (15,810 g) for 20 min. The composition of the unconditioned hydrolysate liquor was determined by high-performance liquid chromatography (HPLC) as described below. The unconditioned hydrolysate liquor contained in g/L: cellobiose, 2.03; glucose, 14.3; xylose, 67.27; arabinose, 11.78; galactose, 7.33; and mannose 5.58, total of 106 g/L.
2.2. Overliming The general protocol used for overliming process is shown in Fig. 1. Target overliming pH maximums of 9, 10, and 11 were evaluated. The detailed protocol was as follows: a 2 L flask was weighed, 1800 mL of hydrolysate
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was added to the flask and it was re-weighed. The flask was placed on heater stir plate and the hydrolysate was heated to 42 8C while stirring using a stir bar. At this time, Ca(OH)2 (Reagent grade, J.T. Baker, USA) was added gradually to the hydrolysate to bring the pH to the target level and the weight of the added lime was recorded. The temperature of the hydrolysate increased to 50–52 8C by addition of the lime, and thereafter was maintained at 50 8C for 30 min using the heater stir plate. Then the mixture was again weighed and filtered at 50 8C using a 0.2 mm filter (Nalgeneunc, Rochester, NY) and the filtrate was allowed to cool to 30 8C. The recovered solids, hereafter referred to as the first cake, were saved for further analysis. The filtrate was then weighed and re-acidified to pH 6 by addition of 10N H2SO4. It was then filter sterilized again using a 0.2 mm filter. This material is hereafter referred to as conditioned liquor. During the reacidification process of the material produced at pH 11 a white precipitate formed. This material was again removed by filtration (0.2 mm), hereafter referred to as the second cake. The amount of acid used and the transient pH changes that occurred after re-acidifying to pH 6 were recorded. A sample of filtrate and second cake from the pH 11-conditioned sample were also saved for elemental analysis. The pH 10 evaluation was repeated for quality control, and samples from the repeat experiment were analyzed in duplicate to check measurement reproducibility.
2.3. Microorganisms and media Zymomonas mobilis 8b, a glucose–xylose fermenting strain [24], was used to evaluate the detoxification efficacy. Stock cultures of this bacterium were stored at 70 8C in cryovials containing RMGX (20 g/L G:10 g/L X) and 20% (v/v) glycerol. Rich medium (RM) (10 g/L yeast extract and 2 g/L KH2PO4) [25] was used as the fermentation nutrient source and was supplemented with hydrolysate (80%, v/v) or reagent grade glucose (G) and xylose (X) to achieve the desired sugar levels.
2.4. Pre-inoculum and inoculum preparation Pre-inoculum was started by thawing a vial of frozen stock culture and transferring its contents into 10 mL RMGX (20 g/L G:10 g/L X) in a 15 mL Falcon sterile tube (Becton Dickinson, Franklin Lakes, NJ.) and incubating for 8 h at 30 8C. The revived culture was inoculated into a 250 mL baffled shake flask containing 200 mL RMGX (40 g/L G:20 g/L X) to achieve an optical density (OD, 600 nm) of 0.05 (0.015 g dry cell mass (DCM)/L). The flask was incubated at 37 8C on a shaker (Innova 4000, New Brunswick, NJ) at 150 rpm, for 12–16 h until the glucose concentration dropped to around 5 g/L. The inoculum culture was then concentrated by centrifuging at 4800 rpm (4147 g) (Beckman, GS-15R, Germany) for 10 min. The OD of the concentrated cell suspension was measured (Spectronic 601, Milton Roy, Ivyland, PA) and the volume needed to inoculate the fermentation to obtain the desired initial cell mass concentration was calculated.
2.5. Fermentation A set of fermentations was performed to compare the fermentability of hydrolysate produced by overliming to different target pHs. These fermentations were done using overlimed hydrolysates (reacidified liquor) that had been diluted to 80% (v/v), a level equivalent to what would be obtained from a 20% (w/w) solids loading dilute acid pretreatment. In addition to hydrolysate fermentations, a pure sugar fermentation run using sugar and acetate concentrations similar to those present in 80% (v/v) hydrolysate was used to evaluate
Fig. 1. Experimental steps used to condition the hydrolysate.
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xylose utilization in the absence of hydrolysate. Sugar and acetate levels were: 20 g/L glucose and 45 g/L xylose and 5 g/L acetate. Fermentations were carried out in BioStat-Q 1-L fermenters (B. Braun, Allentown, PA) at 300 rpm using a working volume 400 mL. The fermentors were inoculated from concentrated cells to achieve an initial OD of 0.5 (0.15 g DCM/L). The pH was maintained at 6 by titrating with 2N KOH. Temperature was controlled at 37 8C.
2.6. Analysis Sugars including glucose, xylose, mannose, galactose, arabinose and cellobiose were analyzed by HPLC (Hewlett Packard, 1090 series, Wilmington, DE) using a Bio-Rad (Hercules, CA) HPX-87H column. Samples were diluted 1:5 for HPLC analysis. Calcium and sulfur were measured in both the filtrate and the solids in each experimental condition. Huffman laboratories, Golden, Colorado, performed elemental analysis of solids and filtrates. Solids were analyzed for carbon, hydrogen, oxygen, nitrogen, sulfur, carbonate and organic C, sulfur, Ca, and ash. Filtrates were analyzed for calcium, sulfur, total carbon, and total solids. The methods they used are as follows: carbon and hydrogen (ASTM D5373), nitrogen (ASTM D5373 for solids and ASTM D5291 for liquids), oxygen (ASTM D5622), sulfur (ASTM D4239 for solids and ASTM D1558 for liquids), ash (ASTM D513-92B), carbonate carbon (ASTM D51392B), calcium by atomic emission spectroscopy, and sulfate by ion chromatography.
Fig. 2. Sugar losses during conditioning at various target pH values (error bars show range for two replicates at pH 10).
2.7. Toxicity assessment Hydrolysate toxicity was assessed by comparing glucose and xylose consumption and ethanol production during fermentation of conditioned hydrolysates.
2.8. Calculations The fractional amount of each sugar remaining after overliming was calculated as the quotient of the mass of each sugar measured in the reacidified liquor divided by that sugar’s mass in the initial hydrolysate. Sugar loss is quantified as 100% minus the percentage of each sugar remaining. Ethanol yield was calculated by dividing the final ethanol concentration by sum of the initial glucose and xylose concentration multiplied by 0.51 g ethanol/ g sugar, the theoretical maximum yield of ethanol from both glucose and xylose [3,26]. We report the ethanol yield calculation in two ways. First, ‘‘overall yield’’ is calculated based on total glucose and xylose initially present in the hydrolysate before conditioning, which takes into account both sugar degradation and hydrolysate fermentability. Alriksson et al. [10] refer to this as the ‘‘balanced ethanol yield’’. Second, ‘‘fermentation ethanol yield’’ is also calculated based on total glucose and xylose present in the hydrolysate after conditioning, which ignores sugar losses that occur during conditioning. The masses of solid and liquor streams, as identified in Fig. 1, were measured and used to calculate an overall mass balance (i.e., total mass out divided by total mass in). The amount of key elements in each phase along with manufacturer analytical information was used to calculate elemental mass balance closures for Ca and S. Mass of components in each stream was determined from measurements of the streams’ masses and densities, for liquid streams, along with component mass fraction (solids) or concentration (liquid). Calcium and sulfur mass fractions in the cake solids and liquid streams were determined from elemental analysis of these materials as reported above. Calcium in the lime was calculated from the weight fractions of Ca(OH)2 and CaCO3 in the reagent, as reported by the manufacturer (98.5% Ca(OH)2 and <0.3% CaCO3). The mass of S in 10N H2SO4 was calculated from the H2SO4 concentration and solution density.
(second cake) formed as pH approached neutrality. This precipitate was analyzed and determined to be primarily gypsum (dehydrated calcium sulfate). No measurable white precipitate was formed during reacidification of hydrolysate overlimed to target pHs of 9 or 10. Fig. 2 shows the sugar (glucose, xylose and arabinose) losses during overliming carried out to target pHs of 9, 10 and 11. Losses of all sugars increase with increasing overliming pH. Losses of xylose, the dominant sugar in the hydrolysate, increase from 7% at pH 9 to 34% at pH 11. Since the concentration of the non-xylose (minor) sugars is low, measurement variability alone introduces significant uncertainty in loss calculations for these sugars. For this reason, the
3. Results During the overliming process, after 30 min of incubation at 50 8C the pH value dropped around 0.5 pH units below the target value. The samples overlimed to pH 11 exhibited unique behavior in that during reacidification a white precipitate
Fig. 3. Percent sugar conversion and ethanol yield for strain Z. mobilis 8b grown on overlimed hydrolysate conditioned at different pHs. Glucose was 100% utilized at all conditions.
A. Mohagheghi et al. / Process Biochemistry 41 (2006) 1806–1811 Table 1 Stream masses (see Fig. 1) and overall mass balance closure pH Mass in (g)
Mass out (g)
Hydrolysate Lime Acid Reacidified First liquor cake 9 10 11 a
1926.0 1920.3 1964.9
34.8 39.2 52.2
6.8 13.9 41.2
1858.1 1851.3 1885.1
Table 3 Percent elemental composition of the first cake from pH 10 (avg.) experiment % Closurea
Component
Composition (%)
99.0 98.9 97.1
Moisture Carbon Hydrogen Oxygen Nitrogen Sulfur Calcium Closure
40.20 (0.0) 9.45 (0.05) 3.25 (0.05) 48.30 (0.4) 0.30 (0.0) 11.7 (0.1) 18.10 (0.0) 91.10 (0.6)
Second cake
89.7 0.0 100.8 0.0 90.8 23.1
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% closure is mass out divided by mass in times 100.
xylose results are believed to be the most accurate, and will be the focus of the subsequent discussion here. The pH 10 conditioning was repeated for quality control purposes and similar results were obtained with 0.5% variation between duplicates for xylose losses. Glucose was utilized completely in all cases but only 75% of the xylose was utilized after overliming to a pH of 10 or 11, and xylose utilization fell to 64% when overliming to pH 9. Fig. 3 shows the xylose utilization and maximum ethanol yield calculated based on initial sugar concentrations in both conditioned (after overliming) and unconditioned (before overliming) hydrolysate. In our previous study [24], we showed that the Z. mobilis strain 8b is not able to ferment neutralized (non-overlimed) hydrolysate under conditions otherwise similar to those used in this study. The results reported here show the effectiveness of overliming for improving hydrolysate fermentability. The results of the overall mass balance closures are shown in Table 1 and in all cases mass balances closed to between 97 and 99%. The slight bias to less than complete closure suggests that a small amount of mass may be lost due to evaporation or incomplete recovery from glassware and filters. These good results confirmed our ability to accurately measure the stream mass flows shown in Fig. 1, which eliminates these measurements as a major source of error in the Ca and S mass balance calculations. Some calcium is present in the feedstock, and sulfur in the form of sulfuric acid is added for pretreatment. As a consequence, both calcium and sulfur are present in the initial hydrolysate. Coupling the calcium and sulfur measurements
with known additions of lime and sulfuric acid upon overliming and reacidification allowed overall calcium and sulfur balances to be calculated. The concentration of calcium and sulfur in the initial hydrolysate is 0.40 and 5.8 g/kg, respectively. Table 2a and b shows summary of calcium and sulfur balance closures obtained during the course of this work. The average is shown for pH 10 condition with the range indicated in parentheses. The results of the elemental analysis of the first cake recovered from the pH 10 experiments are listed in Table 3. This information allowed the gypsum fraction of the cake to be calculated assuming that all of the calcium and sulfur in the precipitate are in the form of gypsum; however, the estimated value changed depending upon whether the calculation was based on calcium or sulfur content. The first cake was calculated to be 69–78% gypsum based on calcium and 50– 66% gypsum based on sulfur; the remaining mass was most likely organic matter. Within the limitations of the underlying assumptions and measurement uncertainties, we conclude that the pH 10 gypsum cake’s composition is approximately 2/3 gypsum and 1/3 unknown organic material and mineral salts. 4. Discussion Material balances around overliming were calculated to improve our understanding of the fate of calcium and sulfur during process. The process design cases [21,22] assume that the only sugars lost in the overliming process are in the liquor removed with the wet gypsum (first cake) solids. They also assume that the first cake solids are exclusively gypsum,
Table 2 Calcium and sulfur concentrations in various streams (see Fig. 1) normalized to g/kg hydrolysate and overall mass balance closure pH
In (g/kg) Hydrolysate
2a: Calcium closure 9 0.4 10b 0.4 [0] 11 0.4 2b: Sulfur closure 9 5.81 5.78 [0.6] 10b 11 5.8 a b
% Closurea
Out (g/kg) Lime
Acid
First cake
Second cake
Cond. liquor
9.6 10.88 [0.32] 14.15
0 0 [0] 0
5.24 5.78 [0.34] 4.17
0 0 [0] 2.03
3.74 3.9 [0.81] 4.63
89.8 85.8 [7.6] 74.4
0.47 0.94 [0.28] 2.65
3.74 3.85 [0.1] 2.44
0 0 [0] 1.83
2.02 1.72 [0.63] 2.03
91.7 82.9 [6.3] 74.6
0 0 [0] 0
% closure is mass out divided by mass in times 100. Average and range (values in parenthesis) of duplicated results.
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although some water and soluble components are carried out with the dewatered gypsum due to incomplete drying following gypsum recovery by solid–liquid separation. The process design cases do not consider that remaining soluble gypsum could detrimentally affect downstream unit operations (e.g., distillation preheaters). The data generated in this work show that these assumptions are not accurate. The calcium, sulfur, and sugar balance data represented here can be used to improve future process models. The best indicator of overliming effectiveness as a hydrolysate conditioning method is the overall ethanol yield, i.e., the yield based on the original sugar content of the unconditioned hydrolysate. As the Fig. 3 shows, the overall ethanol yield was highest (approximately 70%) at pH 10 while the highest sugar recovery was obtained at pH 9. Even though fermentability was highest at pH 11, the higher sugar losses obtained when conditioning at pH 11 lowered the overall ethanol yield for the pH 11 condition compared to the pH 10 condition. In other words, the best yield was achieved at a compromise condition in between the conditions that maximized fermentabilty and those that minimized sugar loss. Sugar loss is conversion of sugars to unfermentable compounds and is known to be a potentially important issue with overliming. Millati et al. [20] reported glucose losses greater than 70% when overliming was carried out for a very long time (170 h) at 60 8C and pH 12. However, sugar losses were low (2%) for treatment times of 1 h or less at pH 10. We obtained much higher sugar loss using a considerably shorter treatment time (30 min). The highest sugar loss was at pH 11 where losses were 14% for glucose, 34% for xylose and 21% for arabinose. Our sugar loss results highlight the importance of keeping the target overliming pH as low as possible to maximize sugar recovery. The reason for the lower sugar losses observed by Millati et al. [20] could be due to the different biomass sugar profile or lower concentrations of sugars in softwood (spruce) hydrolysates (total sugar concentration of 50 g/L) they tested relative to the hydrolysate (total sugar concentration of 106 g/L) tested in our work. The fate of calcium and sulfur in the overliming process has not been studied previously. Calcium is added in the form of lime to increase the pH and a significant portion of it is removed in the gypsum cake after overliming. The remaining calcium is soluble, but at pH 11 additional calcium was precipitated during reacidification. In the pH 9 and 10 cases, approximately 50% of the calcium was removed in the first cake with approximately 40% remaining in the liquor. The unaccounted for 10% or so reflects inaccuracies in the analysis. The overall closures on calcium were 90 and 86% when overliming to pH 9 and 10, respectively. However, when overliming to pH 11 less calcium was present in the first cake (29%) and 14% showed up in the second cake; only 75% of the calcium was accounted for at this condition. Since a significant portion (30–40%) of the calcium remains soluble its effect as a scaling agent in the process should be further investigated. The solubility of calcium in hydrolysate also needs to be compared to its solubility in water and to electrolyte model calculations. If the solubility in hydrolysate
and water are similar, the downstream effects of calcium can be readily estimated using existing process models. In the case of sulfur, it is removed from the initial hydrolysate upon addition of lime and precipitation as gypsum although additional sulfur is then introduced during reacidification. When conditioning to pH 11, some of the additional sulfur added when reacidifying is removed in the second cake. When conditioning to pH 9 or 10, approximately 60% of the sulfur was recovered in the first cake and about 30% remained in the conditioned liquor. The 10% unaccounted for in both cases most likely reflects measurement errors. At pH 11, 29% of the sulfur was recovered in the first cake, 22% in second cake, and only 24% remained in the conditioned liquor. However, in this case, the total closure on sulfur was also only about 75%. Since similar ASTM protocols were used for all the measurements, the low calcium and sulfur closure at pH 11 compared to pH 9 and 10 could be due to limitations of the ASTM protocols for the pH 11 samples. 5. Conclusion Sugar losses increase significantly as overliming pH is raised from 9 to 11. Xylose losses as high as (34%) occurred at pH 11. Conditioning at pH 10 enabled the highest overall ethanol yield to be achieved. The best yield based on sugars present in the unconditioned hydrolysate was achieved at a compromise condition in between the conditions that maximized fermentabilty and those that minimized sugar loss. These results show the importance of pH on overliming process efficacy. The data generated in this work show that previous simplifying assumptions made about the process are inaccurate. Results of the elemental balances on calcium and sulfur closed to 75–96 and 75–92%, respectively, and indicate that approximately 50% of the calcium is contained in the precipitated solids (first cake), with the precipitate being approximately 2/3 gypsum by mass (dry basis). Significant amounts of calcium sulfate remain in the overlimed and reacidified hydrolysate and the impact of this soluble gypsum on downstream operations warrants further investigation. References [1] Ranatunga T, Jervis J, Helm R, McMillan JD, Hatzis C. Identification of inhibitory components toxic toward Zymomonas mobilis CP4(pZB5) xylose fermentation. Appl Biochem Biotechnol 1997;67:185–98. [2] Larsson S, Reimann A, Nilvebrant N, Jonsson L. Comparison of different methods for the detoxification of lignocellulose hydrolysates of spuruce. Appl Biochem Biotechnol 1999;77–79:91–103. [3] McMillan JD. Conversion of hemicellulose hydrolyzates to ethanol. Enzymatic Conversion of Biomass for Fuels Production. In: Himmel M, Baker JO, Overend R, editors. ACS Symposium Series, vol. 566. 1994.p. 411–37. [4] Olsson L, Hahn-Hagerdal B. Fermentation of lignocellulosic hydrolysates for ethanol production. Enz Microb Technol 1996;18:312–31. [5] Leonard RH, Hajny GI. Fermentation of wood sugars to ethyl alcohol. Ind Eng Chem 1945;37:390–5. [6] Van Zyl C, Prior BA, du Preez JC. Production of ethanol from sugar cane bagasse hemicellulose hydrolysate by Pichia stipitis. Appl Biochem Biotechnol 1988;17:357–69.
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Conditioning hemicellulose hydrolysates for fermentation: Effects of overliming pH on sugar and ethanol yields Ali Mohagheghi *, Mark Ruth, Daniel J. Schell National Renewable Energy Laboratory, National Bioenergy Center, 1617 Cole Blvd., Golden, CO 80401, USA Received 19 January 2006; received in revised form 21 March 2006; accepted 24 March 2006
Abstract Overliming is an effective way of conditioning to reduce the toxicity of hydrolysates generated from pretreatment of lignicellulosic biomass for ethanol production. In this work, a range of target overliming pH values from 9 to 11 was studied, and xylose fermenting Zymomonas mobilis strain 8b was used to evaluate the fermentability of overlimed corn stover hemicellulose hydrolysate. pH 11 overlimed hydrolysate was highly fermentable, but xylose losses were the greatest at this condition. Based on ethanol yield and fermentative xylose conversion, pH 10-conditioned hydrolysate produced the best results, 75% xylose utilization and 76% ethanol yield. This condition also produces the highest overall ethanol yield based on total sugars available in the unconditioned hydrolysate, 70%. Overall mass balance closures were very good averaging between 97 and 100% for all experiments. Calcium and sulfur mass balance closures ranged from 75 to 90% and indicated that approximately 50% of the calcium ends up in the gypsum cake, which is calculated to be approximately 63% gypsum. Overall result shows that the pH of the overliming process is the key factor for improving hydrolysate fermentability but too high a pH destroys some of the available sugars and reduces overall ethanol yield. Thus, it is critical to keep the overliming pH as low as possible while making the hydrolysate fermentable. # 2006 Elsevier Ltd. All rights reserved. Keywords: Lime; Gypsum; Conditioning; Corn stover; Zymomonas mobilis; Mass balance
1. Introduction In the context of enzymatically converting lignocellulosic biomass to ethanol utilizing a dilute acid pretreatment step to produce xylose-rich liquor and to enhance cellulose digestibility, a conditioning step is typically required to improve hydrolysate fermentability [1,2]. Many conditioning processes involving chemical, physical and biological methods have been proposed to reduce hydrolysate toxicity prior to fermentation [2–4]. These processes include pH adjustment with lime (calcium oxide) and other caustics [4–10], removal of volatiles using steam stripping and other processes [11], ion exchange [7,12], extraction [12], and adsorption [7,13]. The process most widely used for hydrolysate conditioning is treatment with lime [9,14–17], a process hereafter referred to as ‘‘overliming’’. A study by Larsson (1999) [2] found that overliming is the most cost effective method for detoxifying soft wood hydrolysates. The overliming process begins by
* Corresponding author. Tel.: +1 303 384 6175; fax: +1 303 384 6877. E-mail address: [email protected] (A. Mohagheghi). 1359-5113/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2006.03.028
adding lime to adjust the pH of the hydrolysate liquor to a high value, typically in the range of 9–11. The liquor is usually heated to a temperature in the range of 50–60 8C. Even though lime hydration is a highly exothermic reaction, heating may still be required to reach the desired temperature. Once the pH and temperature are at the target values, the solution is held at temperature for a period of time (typically around 30 min), then filtered to remove the precipitate gypsum (calcium sulfate) formed by divalent calcium from the lime combining with sulfate in the hydrolysate. Finally, re-acidifying the hydrolysate to a value appropriate for fermentation completes the process. The mechanism by which overliming reduces hydrolysate toxicity is not well understood. Agblevor et al. [18] used 13C NMR to determine that overliming removes aliphatic and aromatic acid and other aromatic and aliphatic compounds. The exothermicity of lime hydration and the heating of the hydrolysate during the process decrease the solubility of gypsum and helps to strip off volatile compounds such as furfural [4,19]. A potential drawback of overliming is sugar degradation due to hydroxide-catalyzed degradation reactions; however, overliming to a higher pH produces less toxic (more fermentable) liquor [20].
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A conceptual design was developed at the National Renewable Energy Laboratory for converting corn stover to ethanol using dilute acid prehydrolysis, enzymatic cellulose saccharification, and co-fermentation of biomass sugars [21,22]. Material and energy balances were modeled assuming a 30% solids loading in pretreatment and 20% initial total solids loading into enzymatic saccharification. Overliming conditions and effects were not well understood when the process model was developed but it was assumed that in the overliming step, all of the sulfuric acid in the hydrolysate reacted with lime to produce gypsum. All gypsum was assumed to be insoluble and removed by solid/liquid separation prior to enzymatic hydrolysis and fermentation. The reactions between the acid and caustic streams and the hydrolysate liquor did not consider potential sugar losses during overliming. Also, process model did not take into account that any soluble gypsum could affect other downstream unit operations (e.g., distillation preheaters). The purpose of this study was three-fold. First, to determine how conditioning pH over a range of values from 9 to 11 impacts sugar degradation. Second, to determine the overliming pH that enables the highest ethanol yield. Third, to perform overall mass and calcium and sulfur elemental balances to quantify the partitioning of these elements during the overliming process. While we fixed the overliming temperature and hold time and thus did not attempt to optimize the conditioning step, we sought to better understand the likely features and tradeoffs encountered in a potential industrial process.
2. Materials and methods 2.1. Hydrolysate The hemicellulose hydrolysate used in this work was produced from corn stover using a dilute sulfuric acid pretreatment process carried out in a 1 tonnes/ d pilot-scale continuous reactor operating at a solids concentration of 25% (w/ w), temperature of 190 8C, and an approximate residence time of 1 min, as reported previously [23]. Pretreatment liquor was produced by centrifuging (Sorvall Evolution, Kendro, Asheville, NC) the slurry at 8500 rpm (15,810 g) for 20 min. The composition of the unconditioned hydrolysate liquor was determined by high-performance liquid chromatography (HPLC) as described below. The unconditioned hydrolysate liquor contained in g/L: cellobiose, 2.03; glucose, 14.3; xylose, 67.27; arabinose, 11.78; galactose, 7.33; and mannose 5.58, total of 106 g/L.
2.2. Overliming The general protocol used for overliming process is shown in Fig. 1. Target overliming pH maximums of 9, 10, and 11 were evaluated. The detailed protocol was as follows: a 2 L flask was weighed, 1800 mL of hydrolysate
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was added to the flask and it was re-weighed. The flask was placed on heater stir plate and the hydrolysate was heated to 42 8C while stirring using a stir bar. At this time, Ca(OH)2 (Reagent grade, J.T. Baker, USA) was added gradually to the hydrolysate to bring the pH to the target level and the weight of the added lime was recorded. The temperature of the hydrolysate increased to 50–52 8C by addition of the lime, and thereafter was maintained at 50 8C for 30 min using the heater stir plate. Then the mixture was again weighed and filtered at 50 8C using a 0.2 mm filter (Nalgeneunc, Rochester, NY) and the filtrate was allowed to cool to 30 8C. The recovered solids, hereafter referred to as the first cake, were saved for further analysis. The filtrate was then weighed and re-acidified to pH 6 by addition of 10N H2SO4. It was then filter sterilized again using a 0.2 mm filter. This material is hereafter referred to as conditioned liquor. During the reacidification process of the material produced at pH 11 a white precipitate formed. This material was again removed by filtration (0.2 mm), hereafter referred to as the second cake. The amount of acid used and the transient pH changes that occurred after re-acidifying to pH 6 were recorded. A sample of filtrate and second cake from the pH 11-conditioned sample were also saved for elemental analysis. The pH 10 evaluation was repeated for quality control, and samples from the repeat experiment were analyzed in duplicate to check measurement reproducibility.
2.3. Microorganisms and media Zymomonas mobilis 8b, a glucose–xylose fermenting strain [24], was used to evaluate the detoxification efficacy. Stock cultures of this bacterium were stored at 70 8C in cryovials containing RMGX (20 g/L G:10 g/L X) and 20% (v/v) glycerol. Rich medium (RM) (10 g/L yeast extract and 2 g/L KH2PO4) [25] was used as the fermentation nutrient source and was supplemented with hydrolysate (80%, v/v) or reagent grade glucose (G) and xylose (X) to achieve the desired sugar levels.
2.4. Pre-inoculum and inoculum preparation Pre-inoculum was started by thawing a vial of frozen stock culture and transferring its contents into 10 mL RMGX (20 g/L G:10 g/L X) in a 15 mL Falcon sterile tube (Becton Dickinson, Franklin Lakes, NJ.) and incubating for 8 h at 30 8C. The revived culture was inoculated into a 250 mL baffled shake flask containing 200 mL RMGX (40 g/L G:20 g/L X) to achieve an optical density (OD, 600 nm) of 0.05 (0.015 g dry cell mass (DCM)/L). The flask was incubated at 37 8C on a shaker (Innova 4000, New Brunswick, NJ) at 150 rpm, for 12–16 h until the glucose concentration dropped to around 5 g/L. The inoculum culture was then concentrated by centrifuging at 4800 rpm (4147 g) (Beckman, GS-15R, Germany) for 10 min. The OD of the concentrated cell suspension was measured (Spectronic 601, Milton Roy, Ivyland, PA) and the volume needed to inoculate the fermentation to obtain the desired initial cell mass concentration was calculated.
2.5. Fermentation A set of fermentations was performed to compare the fermentability of hydrolysate produced by overliming to different target pHs. These fermentations were done using overlimed hydrolysates (reacidified liquor) that had been diluted to 80% (v/v), a level equivalent to what would be obtained from a 20% (w/w) solids loading dilute acid pretreatment. In addition to hydrolysate fermentations, a pure sugar fermentation run using sugar and acetate concentrations similar to those present in 80% (v/v) hydrolysate was used to evaluate
Fig. 1. Experimental steps used to condition the hydrolysate.
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xylose utilization in the absence of hydrolysate. Sugar and acetate levels were: 20 g/L glucose and 45 g/L xylose and 5 g/L acetate. Fermentations were carried out in BioStat-Q 1-L fermenters (B. Braun, Allentown, PA) at 300 rpm using a working volume 400 mL. The fermentors were inoculated from concentrated cells to achieve an initial OD of 0.5 (0.15 g DCM/L). The pH was maintained at 6 by titrating with 2N KOH. Temperature was controlled at 37 8C.
2.6. Analysis Sugars including glucose, xylose, mannose, galactose, arabinose and cellobiose were analyzed by HPLC (Hewlett Packard, 1090 series, Wilmington, DE) using a Bio-Rad (Hercules, CA) HPX-87H column. Samples were diluted 1:5 for HPLC analysis. Calcium and sulfur were measured in both the filtrate and the solids in each experimental condition. Huffman laboratories, Golden, Colorado, performed elemental analysis of solids and filtrates. Solids were analyzed for carbon, hydrogen, oxygen, nitrogen, sulfur, carbonate and organic C, sulfur, Ca, and ash. Filtrates were analyzed for calcium, sulfur, total carbon, and total solids. The methods they used are as follows: carbon and hydrogen (ASTM D5373), nitrogen (ASTM D5373 for solids and ASTM D5291 for liquids), oxygen (ASTM D5622), sulfur (ASTM D4239 for solids and ASTM D1558 for liquids), ash (ASTM D513-92B), carbonate carbon (ASTM D51392B), calcium by atomic emission spectroscopy, and sulfate by ion chromatography.
Fig. 2. Sugar losses during conditioning at various target pH values (error bars show range for two replicates at pH 10).
2.7. Toxicity assessment Hydrolysate toxicity was assessed by comparing glucose and xylose consumption and ethanol production during fermentation of conditioned hydrolysates.
2.8. Calculations The fractional amount of each sugar remaining after overliming was calculated as the quotient of the mass of each sugar measured in the reacidified liquor divided by that sugar’s mass in the initial hydrolysate. Sugar loss is quantified as 100% minus the percentage of each sugar remaining. Ethanol yield was calculated by dividing the final ethanol concentration by sum of the initial glucose and xylose concentration multiplied by 0.51 g ethanol/ g sugar, the theoretical maximum yield of ethanol from both glucose and xylose [3,26]. We report the ethanol yield calculation in two ways. First, ‘‘overall yield’’ is calculated based on total glucose and xylose initially present in the hydrolysate before conditioning, which takes into account both sugar degradation and hydrolysate fermentability. Alriksson et al. [10] refer to this as the ‘‘balanced ethanol yield’’. Second, ‘‘fermentation ethanol yield’’ is also calculated based on total glucose and xylose present in the hydrolysate after conditioning, which ignores sugar losses that occur during conditioning. The masses of solid and liquor streams, as identified in Fig. 1, were measured and used to calculate an overall mass balance (i.e., total mass out divided by total mass in). The amount of key elements in each phase along with manufacturer analytical information was used to calculate elemental mass balance closures for Ca and S. Mass of components in each stream was determined from measurements of the streams’ masses and densities, for liquid streams, along with component mass fraction (solids) or concentration (liquid). Calcium and sulfur mass fractions in the cake solids and liquid streams were determined from elemental analysis of these materials as reported above. Calcium in the lime was calculated from the weight fractions of Ca(OH)2 and CaCO3 in the reagent, as reported by the manufacturer (98.5% Ca(OH)2 and <0.3% CaCO3). The mass of S in 10N H2SO4 was calculated from the H2SO4 concentration and solution density.
(second cake) formed as pH approached neutrality. This precipitate was analyzed and determined to be primarily gypsum (dehydrated calcium sulfate). No measurable white precipitate was formed during reacidification of hydrolysate overlimed to target pHs of 9 or 10. Fig. 2 shows the sugar (glucose, xylose and arabinose) losses during overliming carried out to target pHs of 9, 10 and 11. Losses of all sugars increase with increasing overliming pH. Losses of xylose, the dominant sugar in the hydrolysate, increase from 7% at pH 9 to 34% at pH 11. Since the concentration of the non-xylose (minor) sugars is low, measurement variability alone introduces significant uncertainty in loss calculations for these sugars. For this reason, the
3. Results During the overliming process, after 30 min of incubation at 50 8C the pH value dropped around 0.5 pH units below the target value. The samples overlimed to pH 11 exhibited unique behavior in that during reacidification a white precipitate
Fig. 3. Percent sugar conversion and ethanol yield for strain Z. mobilis 8b grown on overlimed hydrolysate conditioned at different pHs. Glucose was 100% utilized at all conditions.
A. Mohagheghi et al. / Process Biochemistry 41 (2006) 1806–1811 Table 1 Stream masses (see Fig. 1) and overall mass balance closure pH Mass in (g)
Mass out (g)
Hydrolysate Lime Acid Reacidified First liquor cake 9 10 11 a
1926.0 1920.3 1964.9
34.8 39.2 52.2
6.8 13.9 41.2
1858.1 1851.3 1885.1
Table 3 Percent elemental composition of the first cake from pH 10 (avg.) experiment % Closurea
Component
Composition (%)
99.0 98.9 97.1
Moisture Carbon Hydrogen Oxygen Nitrogen Sulfur Calcium Closure
40.20 (0.0) 9.45 (0.05) 3.25 (0.05) 48.30 (0.4) 0.30 (0.0) 11.7 (0.1) 18.10 (0.0) 91.10 (0.6)
Second cake
89.7 0.0 100.8 0.0 90.8 23.1
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% closure is mass out divided by mass in times 100.
xylose results are believed to be the most accurate, and will be the focus of the subsequent discussion here. The pH 10 conditioning was repeated for quality control purposes and similar results were obtained with 0.5% variation between duplicates for xylose losses. Glucose was utilized completely in all cases but only 75% of the xylose was utilized after overliming to a pH of 10 or 11, and xylose utilization fell to 64% when overliming to pH 9. Fig. 3 shows the xylose utilization and maximum ethanol yield calculated based on initial sugar concentrations in both conditioned (after overliming) and unconditioned (before overliming) hydrolysate. In our previous study [24], we showed that the Z. mobilis strain 8b is not able to ferment neutralized (non-overlimed) hydrolysate under conditions otherwise similar to those used in this study. The results reported here show the effectiveness of overliming for improving hydrolysate fermentability. The results of the overall mass balance closures are shown in Table 1 and in all cases mass balances closed to between 97 and 99%. The slight bias to less than complete closure suggests that a small amount of mass may be lost due to evaporation or incomplete recovery from glassware and filters. These good results confirmed our ability to accurately measure the stream mass flows shown in Fig. 1, which eliminates these measurements as a major source of error in the Ca and S mass balance calculations. Some calcium is present in the feedstock, and sulfur in the form of sulfuric acid is added for pretreatment. As a consequence, both calcium and sulfur are present in the initial hydrolysate. Coupling the calcium and sulfur measurements
with known additions of lime and sulfuric acid upon overliming and reacidification allowed overall calcium and sulfur balances to be calculated. The concentration of calcium and sulfur in the initial hydrolysate is 0.40 and 5.8 g/kg, respectively. Table 2a and b shows summary of calcium and sulfur balance closures obtained during the course of this work. The average is shown for pH 10 condition with the range indicated in parentheses. The results of the elemental analysis of the first cake recovered from the pH 10 experiments are listed in Table 3. This information allowed the gypsum fraction of the cake to be calculated assuming that all of the calcium and sulfur in the precipitate are in the form of gypsum; however, the estimated value changed depending upon whether the calculation was based on calcium or sulfur content. The first cake was calculated to be 69–78% gypsum based on calcium and 50– 66% gypsum based on sulfur; the remaining mass was most likely organic matter. Within the limitations of the underlying assumptions and measurement uncertainties, we conclude that the pH 10 gypsum cake’s composition is approximately 2/3 gypsum and 1/3 unknown organic material and mineral salts. 4. Discussion Material balances around overliming were calculated to improve our understanding of the fate of calcium and sulfur during process. The process design cases [21,22] assume that the only sugars lost in the overliming process are in the liquor removed with the wet gypsum (first cake) solids. They also assume that the first cake solids are exclusively gypsum,
Table 2 Calcium and sulfur concentrations in various streams (see Fig. 1) normalized to g/kg hydrolysate and overall mass balance closure pH
In (g/kg) Hydrolysate
2a: Calcium closure 9 0.4 10b 0.4 [0] 11 0.4 2b: Sulfur closure 9 5.81 5.78 [0.6] 10b 11 5.8 a b
% Closurea
Out (g/kg) Lime
Acid
First cake
Second cake
Cond. liquor
9.6 10.88 [0.32] 14.15
0 0 [0] 0
5.24 5.78 [0.34] 4.17
0 0 [0] 2.03
3.74 3.9 [0.81] 4.63
89.8 85.8 [7.6] 74.4
0.47 0.94 [0.28] 2.65
3.74 3.85 [0.1] 2.44
0 0 [0] 1.83
2.02 1.72 [0.63] 2.03
91.7 82.9 [6.3] 74.6
0 0 [0] 0
% closure is mass out divided by mass in times 100. Average and range (values in parenthesis) of duplicated results.
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although some water and soluble components are carried out with the dewatered gypsum due to incomplete drying following gypsum recovery by solid–liquid separation. The process design cases do not consider that remaining soluble gypsum could detrimentally affect downstream unit operations (e.g., distillation preheaters). The data generated in this work show that these assumptions are not accurate. The calcium, sulfur, and sugar balance data represented here can be used to improve future process models. The best indicator of overliming effectiveness as a hydrolysate conditioning method is the overall ethanol yield, i.e., the yield based on the original sugar content of the unconditioned hydrolysate. As the Fig. 3 shows, the overall ethanol yield was highest (approximately 70%) at pH 10 while the highest sugar recovery was obtained at pH 9. Even though fermentability was highest at pH 11, the higher sugar losses obtained when conditioning at pH 11 lowered the overall ethanol yield for the pH 11 condition compared to the pH 10 condition. In other words, the best yield was achieved at a compromise condition in between the conditions that maximized fermentabilty and those that minimized sugar loss. Sugar loss is conversion of sugars to unfermentable compounds and is known to be a potentially important issue with overliming. Millati et al. [20] reported glucose losses greater than 70% when overliming was carried out for a very long time (170 h) at 60 8C and pH 12. However, sugar losses were low (2%) for treatment times of 1 h or less at pH 10. We obtained much higher sugar loss using a considerably shorter treatment time (30 min). The highest sugar loss was at pH 11 where losses were 14% for glucose, 34% for xylose and 21% for arabinose. Our sugar loss results highlight the importance of keeping the target overliming pH as low as possible to maximize sugar recovery. The reason for the lower sugar losses observed by Millati et al. [20] could be due to the different biomass sugar profile or lower concentrations of sugars in softwood (spruce) hydrolysates (total sugar concentration of 50 g/L) they tested relative to the hydrolysate (total sugar concentration of 106 g/L) tested in our work. The fate of calcium and sulfur in the overliming process has not been studied previously. Calcium is added in the form of lime to increase the pH and a significant portion of it is removed in the gypsum cake after overliming. The remaining calcium is soluble, but at pH 11 additional calcium was precipitated during reacidification. In the pH 9 and 10 cases, approximately 50% of the calcium was removed in the first cake with approximately 40% remaining in the liquor. The unaccounted for 10% or so reflects inaccuracies in the analysis. The overall closures on calcium were 90 and 86% when overliming to pH 9 and 10, respectively. However, when overliming to pH 11 less calcium was present in the first cake (29%) and 14% showed up in the second cake; only 75% of the calcium was accounted for at this condition. Since a significant portion (30–40%) of the calcium remains soluble its effect as a scaling agent in the process should be further investigated. The solubility of calcium in hydrolysate also needs to be compared to its solubility in water and to electrolyte model calculations. If the solubility in hydrolysate
and water are similar, the downstream effects of calcium can be readily estimated using existing process models. In the case of sulfur, it is removed from the initial hydrolysate upon addition of lime and precipitation as gypsum although additional sulfur is then introduced during reacidification. When conditioning to pH 11, some of the additional sulfur added when reacidifying is removed in the second cake. When conditioning to pH 9 or 10, approximately 60% of the sulfur was recovered in the first cake and about 30% remained in the conditioned liquor. The 10% unaccounted for in both cases most likely reflects measurement errors. At pH 11, 29% of the sulfur was recovered in the first cake, 22% in second cake, and only 24% remained in the conditioned liquor. However, in this case, the total closure on sulfur was also only about 75%. Since similar ASTM protocols were used for all the measurements, the low calcium and sulfur closure at pH 11 compared to pH 9 and 10 could be due to limitations of the ASTM protocols for the pH 11 samples. 5. Conclusion Sugar losses increase significantly as overliming pH is raised from 9 to 11. Xylose losses as high as (34%) occurred at pH 11. Conditioning at pH 10 enabled the highest overall ethanol yield to be achieved. The best yield based on sugars present in the unconditioned hydrolysate was achieved at a compromise condition in between the conditions that maximized fermentabilty and those that minimized sugar loss. These results show the importance of pH on overliming process efficacy. The data generated in this work show that previous simplifying assumptions made about the process are inaccurate. Results of the elemental balances on calcium and sulfur closed to 75–96 and 75–92%, respectively, and indicate that approximately 50% of the calcium is contained in the precipitated solids (first cake), with the precipitate being approximately 2/3 gypsum by mass (dry basis). Significant amounts of calcium sulfate remain in the overlimed and reacidified hydrolysate and the impact of this soluble gypsum on downstream operations warrants further investigation. References [1] Ranatunga T, Jervis J, Helm R, McMillan JD, Hatzis C. Identification of inhibitory components toxic toward Zymomonas mobilis CP4(pZB5) xylose fermentation. Appl Biochem Biotechnol 1997;67:185–98. [2] Larsson S, Reimann A, Nilvebrant N, Jonsson L. Comparison of different methods for the detoxification of lignocellulose hydrolysates of spuruce. Appl Biochem Biotechnol 1999;77–79:91–103. [3] McMillan JD. Conversion of hemicellulose hydrolyzates to ethanol. Enzymatic Conversion of Biomass for Fuels Production. In: Himmel M, Baker JO, Overend R, editors. ACS Symposium Series, vol. 566. 1994.p. 411–37. [4] Olsson L, Hahn-Hagerdal B. Fermentation of lignocellulosic hydrolysates for ethanol production. Enz Microb Technol 1996;18:312–31. [5] Leonard RH, Hajny GI. Fermentation of wood sugars to ethyl alcohol. Ind Eng Chem 1945;37:390–5. [6] Van Zyl C, Prior BA, du Preez JC. Production of ethanol from sugar cane bagasse hemicellulose hydrolysate by Pichia stipitis. Appl Biochem Biotechnol 1988;17:357–69.
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