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Effect of pH, time and temperature of overliming on detoxification of dilute-acid hydrolyzates for fermentation by Saccharomyces cerevisiae
Process Biochemistry 38 (2002) 515 /522 www.elsevier.com/locate/procbio
Effect of pH, time and temperature of overliming on detoxification of dilute-acid hydrolyzates for fermentation by Saccharomyces cerevisiae Ria Millati *, Claes Niklasson, Mohammad J. Taherzadeh Department of Chemical Reaction Engineering, Chalmers University of Technology, S-41296 Goteborg, Sweden Received 4 February 2002; accepted 12 May 2002
Abstract The effects of different variables in detoxification of a severely inhibiting dilute-acid hydrolyzate by overliming were investigated. Overliming was carried out by increasing the pH to 10, 11 or 12 at two different temperatures, 25 and 60 8C, holding the pH and temperature at constant values for different periods of time, 0, 1, 20 and 170 h, and then adjusting the pH to 5.5. All hydrolyzates were then fermented in batch cultivation by Saccharomyces cerevisiae in shake flasks, whereupon one was then selected for continuous cultivation in a bioreactor. The most significant effect of overliming was a sharp decrease in the concentration of furfural and hydroxymethylfurfural, whereas the concentration of acetic acid remained unchanged and the decrease in the total phenolic compounds was less than 30%. Detoxification at pH 12 for more than 1 h was effective, whereas no effect was obtained at pH 10 and the hydrolyzates had to remain at pH 11 for more than 20 h to become fermentable. On the other hand, decrease in sugar concentration during overliming was a serious problem at pH 12, especially at the higher temperature, where up to 70% sugars were degraded. The fermentability of a detoxified hydrolyzate was also tested in a continuous cultivation by immobilized S. cerevisiae in Ca-alginate. The hydrolyzate was fully fermentable at different dilution rates between 0.2 and 1.0 h 1. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Detoxification; Saccharomyces cerevisiae ; Hydrolyzates; Overliming; Fermentation; Immobilization
1. Introduction Ethanol from renewable resources has been of interest in recent decades as an alternative fuel or oxygenate additive to the current fossil fuels. Lignocellulosic materials are cheap renewable resources, available in large amounts. In this process, hydrolysis should first be carried out to open the crystalline structure of the lignocellulose and decompose the cellulose and hemicellulose polymers to their monomer sugars. The sugars are then fermented to ethanol by e.g. Baker’s yeast. A number of undesirable by-products are formed during the hydrolysis, which inhibit the fermentation process. The most important by-products in terms of concentration in dilute-acid hydrolysis were identified as furan derivatives, e.g. furfural and 5-hydroxymethylfurfural
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(HMF), carboxylic acids, e.g. acetic acid, formic acid, and levulinic acid, and phenolic compounds [1]. Cultivation of the hydrolyzates can be carried out in different modes of operation including batch, fed-batch and continuous fermentation. The fermentation of hydrolyzates has traditionally been investigated in batch processes. Despite its simplicity, there is a major drawback of the batch fermentation, which is the high initial concentration of the inhibitors in the medium. It results in a long lag phase or sometimes even complete failure of the fermentation [2,3]. The fed-batch cultivation is of interest to overcome some of these problems [4,5]. This is due to the capability of the yeast for in situ detoxification of the hydrolyzates and conversion of the inhibitors such as furfural and HMF to some less toxic compounds [6 /8]. The capability of in situ detoxification of the cells makes continuous cultivation an attractive method for the fermentation of dilute-acid hydrolyzates. However, the dilution rate in continuous cultivation would be a function of the inhibitor concentrations, i.e. a lower
0032-9592/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 2 - 9 5 9 2 ( 0 2 ) 0 0 1 7 6 - 0
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dilution rate should be applied for a feed with higher concentration of inhibitors [9]. Furthermore, if a very low dilution rate is applied to ferment severely inhibiting hydrolyzates, the conversion rate of the inhibitors decreases with the decreased growth of the biomass [7,8]. This results in wash out of the cells from the bioreactor, even at very low dilution rates. Washout can be avoided, if the biomass is retained by e.g. filtration, recirculation or immobilization of the biomass. In a previous study [10], the continuous cultivation by immobilized Saccharomyces cerevisiae was shown to be able to ferment dilute-acid hydrolyzates with a high ethanol production rate. However, when the cells face a severely inhibiting hydrolyzate, metabolic activity and ethanol productivity decreased to zero, in spite of the presence of high cell mass concentration in the beads. Detoxification prior to fermentation is probably necessary to retain metabolically ‘active’ biomass, when a toxic hydrolyzate is used as feed. There are several detoxification methods including biological treatments, e.g. laccase and Trichoderma reesei , physical treatments, e.g. extraction, and chemical treatments, e.g. ion exchange, evaporation, overliming and sulphite treatment [11 /18]. Because of its low costs and high efficiency among the detoxification methods, overliming has been recognized as the most common treatment, although it has a drawback in terms of sugar losses [15]. Overliming is traditionally performed by the addition of alkali (Ca(OH)2 or NaOH) to increase the pH up to 10, followed by an adjustment to the cultivation pH [11,18]. The aim of the current work was to investigate the capability of overliming in detoxification and in increasing the fermentability of dilute-acid hydrolyzates in batch and continuous modes of operation. The effects of three variables in overliming were examined: pH, duration and temperature of detoxification. The fermentability of the detoxified hydrolyzates was examined by batch cultivation in shake flasks, and a selected hydrolyzate was further tested by continuous cultivation of immobilized S. cerevisiae in a bioreactor.
The pH of the hydrolyzates was less than 2. Leaving the hydrolyzate at room temperature for a week resulted in an average of 10% decrease in sugar concentration. 2.2. Overliming treatment The detoxification by the overliming procedure involved increasing the pH of the hydrolyzates to 10.0, 11.0 or 12.0 by addition of Ca(OH)2. The pH was held at the increased level for different periods of time 0, 1, 20 and 170 h, at two different temperatures, 25 and 60 8C. The pH of the detoxified hydrolyzates was then adjusted to the cultivation pH, followed by sterilization with autoclave, and the media were inoculated immediately. 2.3. Yeast strain and medium The yeast S. cerevisiae CBS 8066, obtained from the Central bureau voor Schimmel cultures (Delft, The Netherlands) was used in all experiments. The strain was maintained on agar plates made from yeast extract 10 g/l, soy peptone 20 g/l, and agar 20 g/l with D-glucose 20 g/l as an additional carbon source. Inoculum cultures were grown in 300-ml cotton-plugged-conical flasks on a shaker at 30 8C for 24 h. The liquid volume was 100 ml. The growth medium was a defined synthetic medium including 50 g/l glucose as previously reported [19]. 2.4. Immobilization procedure A 100-ml inoculum culture was mixed with 400 ml sterilized sodium alginate to give a final concentration of alginate solution of 3% (w/v). The beads were formed by slowly dropping the alginate solution into 1 l of gently stirred calcium chloride solution (30 g/l). The beads diameter formed through a small opening tube were between 3 and 4 mm [10]. The complete hardening of beads was achieved overnight. 2.5. Cultivation in shake flasks
2. Material and methods 2.1. Dilute-acid hydrolyzates The hydrolyzate used in the experiments was produced from forest residuals originated mainly from spruce [10]. The chips were hydrolyzed by 0.5% H2SO4 at 12/21 bar in a 350-l rebuilt masonite gun batch reactor in two stages. This has been described previously [10]. The hydrolyzates of the two stages were then mixed and stored at 4 8C before use. The hydrolyzate contained 20.0 g/l glucose, 18.1 g/l mannose, 4.2 g/l galactose, 7.8 g/l xylose, 1.95 g/l HMF, 0.8 g/l furfural, 3.0 g/l acetic acid and 2.4 g/l total phenolic compounds.
Cultivation in shake flasks was performed to test the fermentability of the detoxified hydrolyzates by overliming at different conditions. The detoxified hydrolyzates were pH-readjusted to 5.5 and then added to the culture medium, which was a defined synthetic medium with no carbon source similar to the inoculum. The cultivations were carried out in 300-ml cotton-pluggedErlenmeyer flasks placed in a shaker bath at a temperature of 30 8C for 48 h without pH controlling. The total liquid volume was 155 ml including 135-ml hydrolyzates, 5 ml inoculum, and 15 ml of a solution containing the other minerals and vitamins. Liquid samples were withdrawn at 0, 2, 5, 8, 24 and 48 h, which were analyzed by HPLC. Similar experiments were also
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carried out with undetoxified hydrolyzates. All the experiments were duplicated. 2.6. Continuous cultivation with immobilized cells Continuous cultivations were carried out in a BiostatA bioreactor (B. Braun Biotech International, Germany) at a temperature of 30 8C and stirring rate of 500 rpm with a working volume of 1.0 l. A detoxified hydrolyzate was fed into the bioreactor with a peristaltic pump (Watson-Marlow Alitea AB, Sweden) without pH re-adjustment. The pH value in the medium was controlled at 5.0 (9/0.07) by addition of 3 M HCl. Anaerobic conditions were maintained by continuous nitrogen sparging at a flow rate of 0.8 l/min controlled by a Hi-Tech mass flow controller (Ruurlo, The Netherlands). The cultivation was started in a batch mode in a fully defined medium [19] with glucose concentration of 50 g/l to prepare a high concentration of cells inside the beads. The hydrolyzates and the entire mineral and vitamins were then fed separately to the bioreactor at certain dilution rates. 2.7. Analytical methods The carbon dioxide and oxygen content in the outlet gas of the bioreactor were continuously measured with an acoustic gas monitor (model 1311, Innova, Denmark). Samples for HPLC analysis were withdrawn from the shake flasks or bioreactor, centrifuged and stored at /20 8C before analyzing. Glucose, xylose, galactose and mannose were analyzed on an Aminex HPX-87P column (Bio-Rad, USA) at 85 8C. Ultra-pure water was used as eluent at a flow rate of 0.6 ml/min. Ethanol, acetic acid, lactic acid, glycerol, furfural and HMF concentrations were determined by an Aminex HPX-87H column (Bio-Rad) at 60 8C with 0.6 ml/min eluent of 5 mM H2SO4. A refractive index (RI) detector (Waters 410, Millipore, Milford, USA) and an UV absorbance detector (Waters 486) were used in series. Concentrations of all mentioned metabolites were determined from the RI chromatograms, except lactic acid, furfural and HMF, which were determined from the UV chromatograms at 210 nm. The standard deviation of analyses by the HPLC was 3%. Total phenols concentration was analyzed by a spectrophotometric method with Folin & Ciocalteaus reagent at absorbance 725 nm [20].
3. Results 3.1. Detoxification The effect of three different parameters in detoxification by overliming with Ca(OH)2 was investigated:
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detoxification pH (pHd), time for detoxification (td) and temperature (Td). The detoxification was carried out by adjusting the temperature (Td), increasing the pH from the initial pH (i.e. less than 2) with calcium hydroxide to pHd, holding the pHd and Td unchanged for a period of time (td) and then decreasing the pH to the cultivation pH. These three variables were given as the following values: pHd f10; 11; 12g td f0; 1; 20; 170g (h) Td f25; 60g ( C) The concentration profiles of the sugars including glucose, mannose, galactose and xylose as well as furans (furfural and HMF), total phenolic compounds and acetic acid at different pHd, td and Td are summarized in Table 1 and Table 2. The concentrations of furans were highly affected by both the pHd and td, whereas there was not a clear difference between the furans’ profiles obtained at two different Td. The concentration of both furfural and HMF approached zero, while the pHd increased to 12 at the duration td higher than 20 h. However, the concentration of acetic acid was not generally affected by changing in any of the variables pHd, td or Td. The concentration of total phenolic compounds decreased by less than 30% at different pHd and td at Td /25 8C (Table 1). However, an increase in the phenolic compounds was observed at higher temperature and pHd (Table 2). The results show a general trend of decreasing sugar concentration, while pHd or td is increased. The maximum decrease in glucose concentration was more than 60%, when the hydrolyzate was treated at pHd 12 for td 170 h at 25 8C (Table 1). There was a similar trend for the other sugars, where the concentration of the total sugars was decreased by 68% at these treatment conditions. The missing sugars were generally found in the form of lactic acid (Fig. 1). 3.2. Batch cultivation of detoxified hydrolyzates Batch cultivations of the detoxified hydrolyzates were carried out in shake flasks, in order to examine the performance of the detoxification procedure. A portion of 135 ml of hydrolyzates was added to a total volume of 155 ml of fully synthetic medium containing 0.025 g of dry-weight yeast cells, and cultivated for 48 h. A summary of the most important results is presented in Fig. 2 and Fig. 3. None of the detoxified hydrolyzates at pHd 10 could be fermented, regardless of the treatment duration (td) and temperature (Td). In addition, no consumption of glucose and formation of ethanol were observed during the cultivation of the detoxified hydrolyzates at td /0. However, increasing either of these two variables, i.e.
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Table 1 The composition of hydrolyzates after different treatments of overliming at 25 8C Treatment pH (pHd)
Glucose (g/l)
Mannose (g/l)
Galactose (g/l)
Xylose (g/l)
HMF (g/l)
Furfural (g/l)
Acetic (g/l)
Total phenol (g/l)
0 0 0
10 11 12
20.1 20.1 18.6
18.4 20.1 16.6
4.4 3.5 3.6
7.8 7.7 7
2.03 0.78 1.51
0.40 0.82 0.28
2.8 3.0 2.4
2.5 2.4 2.4
1 1 1
10 11 12
19.8 19.4 17.4
17.9 18.1 15.8
4.1 2.8 3
7.5 7.3 6.4
1.91 1.32 0.49
0.37 0.64 0.06
2.8 2.8 2.5
2.4 2.4 2.2
20 20 20
10 11 12
20.6 19.6 13.5
19.7 20 12.7
3.7 3.3 0.6
7.3 6.6 2
1.43 0.56 0.02
0.27 0.06 0.00
2.7 2.7 2.7
2.3 2.2 2.1
170 170 170
10 11 12
18.5 18.9 10.6
16.5 18.7 9.5
3.1 3.4 1.3
7.5 7.2 1.8
1.19 0.35 0.04
0.29 0.06 0.00
2.5 2.9 2.5
2.4 1.8 2.7
Table 2 The composition of hydrolyzates after different treatments of overliming at 60 8C Treatment duration td (h)
Treatment pH (pHd)
Glucose (g/l)
Mannose (g/l)
Galactose (g/l)
Xylose (g/l)
HMF (g/l)
Furfural (g/l)
Acetic (g/l)
Total phenol (g/l)
0 0 0
10 11 12
19.5 19.4 19.8
17.6 18.4 19.8
3.9 3.2 3.4
7.6 7.3 7.3
1.96 1.89 1.59
0.38 0.89 0.74
2.7 3.1 3.1
2.3 1.7 2.3
1 1 1
10 11 12
19.1 20.0 18.4
17.3 20.1 19.8
3.8 3.5 2.4
7.5 7.4 6.1
1.57 1.39 0.35
0.28 0.63 0.17
2.3 3.0 3.1
2.4 1.8 6.0
20 20 20
10 11 12
20.4 16.1 5.8
20.4 16.9 5.7
3.6 2.9 1.2
7.3 5.4 0.9
1.55 0.08 0.02
0.28 0.02 0.00
2.8 2.8 2.9
2.3 3.0 4.5
170 170 170
10 11 12
18.7 15.5 7.1
17.1 14.2 6.6
3.1 2.7 1.3
7.3 5.7 1.0
1.35 0.27 0.04
0.35 0.05 0.00
2.8 2.7 2.9
2.1 2.6 3.6
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Treatment duration td (h)
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Fig. 1. The concentration profile of the fermentable sugars at different detoxification pHd of 10 (j), 11 (k) and 12 (') and produced lactic acid at pHd of 10 (I), 11 (m) and 12 (^) with various treatment durations (td) at two different Td of (a) 25 8C and (b) 60 8C.
pHd and td, will increase the fermentability. Among the 1 h detoxified hydrolyzates, the only ones with pHd 12 were fermentable, but with a long lag phase (Figs. 2 and 3). The lag phase of the hydrolyzate treated at 25 8C
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was somewhat shorter, since 1.5 g/l ethanol has been produced after 24 h cultivation. The fermentability of the treated hydrolyzates at pHd 11 was dependent on treatment duration, and increased by increasing ‘td’. While the hydrolyzates treated at this pHd for 0 and 1 h were not fermentable at either Td, treatment duration of 20 h at 25 8C made it fermentable but with a long lag phase (Fig. 2). Increasing the td to 170 h, cause the lag phase to be decreased. The treatment at higher temperature (Td /60 8C) resulted in a worse fermentability: at pHd 11 and td of 20 h the hydrolyzates were not fermentable, and by td of 170 h they were fermentable but with a long lag phase. The best fermentability was obtained at pHd 12, although td played an important role in the results. The quickly treated hydrolyzates at pHd 12, resulted in no fermentation within 48 h. However, when td increased to 1 h at either temperature of 25 or 60 8C, the detoxified hydrolyzates were fermentable but with a long lag phase (Figs. 2 and 3). Further increase in td to 20 or 170 h resulted in fully fermentable hydrolyzates. On the other hand, the increased td resulted in decline in the sugar concentration and formation of lactic acid (Fig. 1).
Fig. 2. The profile of ethanol production, glucose consumption and total furans for detoxified hydrolyzates at pHd of 10 (j), 11 (k) and 12 (') and various td of 0, 1, 20 and 170 h at 25 8C.
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Fig. 3. The profile of ethanol production, glucose consumption and total furans for detoxified hydrolyzates at pHd of 10 (j), 11 (k) and 12 (') and various td of 0, 1, 20 and 170 h at 60 8C.
3.3. Continuous cultivation of detoxified hydrolyzates The performance of detoxification was investigated in anaerobic continuous cultivation by immobilized cells of S cerevisiae. The inoculation medium (100 ml) containing 0.5 g dry cells was immobilized in a total volume of 500 ml beads of 3 /4 mm in diameter. The fermentation was started with a batch cultivation at pH 5 in a fully synthetic medium and 50 g/l glucose as carbon and energy source. The cells were grown up in the beads to reach a concentration of approximately 5 g/l. In order to check the fermentability of undetoxified hydrolyzates in continuous cultivation, it was fed to the
Fig. 4. Carbon dioxide evolution rate in continuous cultivation of the detoxified hydrolyzate by immobilized S. cerevisiae at two different dilution rates. The dashed lines show the points where the feed was changed.
bioreactor at a dilution rate (D ) of 0.1 and 0.2 h1 in different experiments. The feeding continued for 40 h, but the cells could not survive in either cases, carbon dioxide evolution rate (CER) declined to zero, and the residual glucose was increased to the level of glucose present in the feed. The continuous cultivation of a detoxified hydrolyzate was carried out similarly to the undetoxified one by addition of the hydrolyzate at dilution rates 0.2, 0.4, 0.6, 0.8 and 1.0 h 1. The selected hydrolyzate was overlimed at pHd 12, td of 20 h, and at Td of 25 8C. The treatment at these conditions was chosen due to its proper fermentability results in the batch cultivation. The cultivations were carried out and the CER was stabilized in all the stages. The cultivation continued at each stage for 5 residence times in order to be sure of achieving steady-state conditions. The feeding stopped between two stages until CER decreased to less than 2% of the CER at the steady-state conditions (e.g. cf. Fig. 4). A summary of the results at each dilution rate is presented in Table 3. The detoxification of the selected hydrolyzate was fully successful, since more than 80% of glucose and 70% of mannose were consumed even at high dilution rates. The residual glucose was zero at the lowest dilution rate and increased to 2.1 g/l at the highest D , i.e. 1.0 h1. The residual furfural and HMF were detected as less than 0.03 g/l in the bioreactors in
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Table 3 The residual sugars, furans and acetic acid in the medium during continuous cultivation of the detoxified hydrolyzate at pHd 12, td 20 h and Td 25 8C by immobilized S. cerevisiae at different dilution rates Concentration (g/l)
D 0.2 h 1
D 0.4 h 1
D 0.6 h 1
D 0.8 h 1
D 1.0 h 1
Residual glucosea Residual mannoseb Residual furfural Residual HMF Acetic acid
0 0.93 0.002 0.008 1.40
0.28 1.49 0.025 0.013 2.31
1.61 3.33 0.025 0.011 2.28
1.69 2.51 0.015 0.016 1.77
2.10 2.19 0.012 0.016 1.52
a b
Glucose concentration in the feed 12.1 g/l. Mannose concentration in the feed 11.4 g/l.
all the experiments. The concentration of acetic acid in the bioreactor was less than the corresponding concentration in the feed (Table 3), which means that part of the acid has been consumed by the yeast.
4. Discussion Detoxification by overliming is a promising method to increase the fermentability of the dilute-acid hydrolyzates. A conventional overliming is usually performed by adding of a base, e.g. Ca(OH)2 up to pH 10 or 11 at 25 or 60 8C, waiting for 30 /60 min and then decreasing the pH to a level suitable for the fermentation [11,15,16,18]. The results of the current work show the importance of choosing the right detoxification pH (pHd), duration (td) and temperature (Td). These variables can seriously affect the results of the detoxification process and the amount of sugars in the hydrolyzates. In general, the effect of these three variables on detoxification can be summarized as: 1) Any increase in pHd will enhance the detoxification and increase the fermentability of the hydrolyzates, but causes a decline in sugar concentration as well. 2) The detoxification process is not a quick process. The treatment duration (td) plays an important role in it, and longer duration increases the fermentability of the hydrolyzates. 3) The effect of temperature Td is not as drastic as the effects of pHd and td. The treatment at lower temperature generally resulted in a better fermentability. The effect of overliming is obvious in decreasing the concentration of furans (Tables 1 and 2), whereas the concentration of acetic acid remains unchanged and the total phenolic compounds decreases just a few percent. Among the phenolic compounds, less heavily substituted phenolics are probably the inhibitoriest materials in the hydrolyzates [21]. Phenol and vanillin are of the strongest phenolic compounds, but they have marginal inhibition effects at concentration of 1 g/l [22]. Although furans are not considered as strong inhibitors in such
low concentrations as appeared in the hydrolyzates [7 / 9], but it seems that the partial removal of furfural and HMF is enough to make the hydrolyzate fermentable. Acetic acid is sometimes considered as inhibitor and sometimes as enhancer for the fermentation [19,23]. Acetic acid would inhibit the fermentation, if the concentration of undissociated acetic acid increases to higher than 5 g/l. It is far from the condition present in the current work. The results of the current work are in the line of previous reports on the effects of overliming [11,16]. However, it should be noticed that the quantitative extrapolation of the values presented in the current or previous works to any other hydrolyzates should be carried out very carefully, since the hydrolysis process is not usually well reproducible. Achieving a better fermentability of the toxic hydrolyzates by overliming is at the cost of a decline in sugar concentration. The sugar degradation has not been so critical up to pHd 11 in a short time, whereas a major part of the sugar can be degraded at higher pHd in a longer td. The degradation of sugars was accompanied by appearance of lactic acid (Fig. 3), which is a known product of sugar decomposition in alkali solution [24,25]. The hexoses are believed to decompose first to 3-desoxy-hexosone, then to glyceraldehydes and methylglyoxal or metasaccharic acid, and then to lactic acid and glyceric aldehyde [24]. It can be concluded that overliming is not a simple process with defined variables. All the studied variables including detoxification pH, duration and temperature, and some other variables such as the type of base used, can affect the degree of detoxification. Since higher pHd or td gives better detoxification but at the cost of sugar depletion, an optimization for each batch of hydrolyzate is perhaps necessary in order to find the best overliming conditions.
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[14] Rivard CJ, Engel RE, Hayward TK, Nagle NJ, Hatzis C, Philippidis GP. Measurement of the inhibitory potential and detoxification of biomass pretreatment hydrolyzate for ethanol production. Appl Biochem Biotechnol 1996;57/58:183 /91 (Seventeenth Symposium on Biotechnology for Fuels and Chemicals, 1995). [15] Martinez A, Rodriguez ME, York SW, Preston JF, Ingram LO. Effects of Ca(OH)2 treatments (‘overliming’) on the composition and toxicity of bagasse hemicellulose hydrolysates. Biotechnol Bioeng 2000;69:526 /36. [16] Martinez A, Rodriguez ME, Wells ML, York SW, Preston JF, Ingram LO. Detoxification of dilute acid hydrolysates of lignocellulose with lime. Biotechnol Prog 2001;17:287 /93. [17] Ranatunga T, Jervis J, Helm R, McMillan J, Wooley R. The effect of overliming on the toxicity of dilute acid pretreated lignocellulosics: the role of inorganics, uronic acids and ethersoluble organics. Enzyme Microb Technol 2000;27:240 /7. [18] Leonard R, Hajny G. Fermentation of wood sugars to ethyl alcohol. Ind Eng Chem 1945;37:390 /5. [19] Taherzadeh MJ, Lide´n G, Gustafsson L, Niklasson C. The effect of pantothenate deficiency and acetate addition on anaerobic batch fermentation of glucose by Saccharomyces cerevisiae . Appl Microbiol Biotechnol 1996;46:176 /82. [20] Nilvebrant NO, Reimann A, Larsson S, Jonsson LJ. Detoxification of lignocellulose hydrolysates with ion-exchange resins. Appl Biochem Biotechnol 2001;91 /93:35 /49. [21] Clark T, Mackie K. Fermentation inhibitors in wood hydrolysates derived from the softwood Pinus radiata . J Chem Tech Biotechnol 1984;34B:101 /10. [22] Pfeifer PA, Bonn G, Bobleter O. Influence of biomass degradation products on the fermentation of glucose to ethanol by Saccharomyces carlsbergensis W 34. Biotechnol Lett 1984;6:541 /6. [23] Taherzadeh MJ, Niklasson C, Lide´n G. Acetic acid */friend or foe in anaerobic batch conversion of glucose to ethanol by Saccharomyces cerevisiae ? Chem Eng Sci 1997;52:2653 /9. [24] Telegdy-Kovats L, Orsi F, Rasky K. Kinetics of lactic acid production during glucose and fructose decomposition in alkaline medium. Nahrung 1970;14:9 /12. [25] Ohashi K, Takahashi T. Utilization of starch. II. Decomposition of glucose in alkaline solutions. Res Bull Gifu Coll Agr (Japan) 1950;68:74 /9.
Effect of pH, time and temperature of overliming on detoxification of dilute-acid hydrolyzates for fermentation by Saccharomyces cerevisiae Ria Millati *, Claes Niklasson, Mohammad J. Taherzadeh Department of Chemical Reaction Engineering, Chalmers University of Technology, S-41296 Goteborg, Sweden Received 4 February 2002; accepted 12 May 2002
Abstract The effects of different variables in detoxification of a severely inhibiting dilute-acid hydrolyzate by overliming were investigated. Overliming was carried out by increasing the pH to 10, 11 or 12 at two different temperatures, 25 and 60 8C, holding the pH and temperature at constant values for different periods of time, 0, 1, 20 and 170 h, and then adjusting the pH to 5.5. All hydrolyzates were then fermented in batch cultivation by Saccharomyces cerevisiae in shake flasks, whereupon one was then selected for continuous cultivation in a bioreactor. The most significant effect of overliming was a sharp decrease in the concentration of furfural and hydroxymethylfurfural, whereas the concentration of acetic acid remained unchanged and the decrease in the total phenolic compounds was less than 30%. Detoxification at pH 12 for more than 1 h was effective, whereas no effect was obtained at pH 10 and the hydrolyzates had to remain at pH 11 for more than 20 h to become fermentable. On the other hand, decrease in sugar concentration during overliming was a serious problem at pH 12, especially at the higher temperature, where up to 70% sugars were degraded. The fermentability of a detoxified hydrolyzate was also tested in a continuous cultivation by immobilized S. cerevisiae in Ca-alginate. The hydrolyzate was fully fermentable at different dilution rates between 0.2 and 1.0 h 1. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Detoxification; Saccharomyces cerevisiae ; Hydrolyzates; Overliming; Fermentation; Immobilization
1. Introduction Ethanol from renewable resources has been of interest in recent decades as an alternative fuel or oxygenate additive to the current fossil fuels. Lignocellulosic materials are cheap renewable resources, available in large amounts. In this process, hydrolysis should first be carried out to open the crystalline structure of the lignocellulose and decompose the cellulose and hemicellulose polymers to their monomer sugars. The sugars are then fermented to ethanol by e.g. Baker’s yeast. A number of undesirable by-products are formed during the hydrolysis, which inhibit the fermentation process. The most important by-products in terms of concentration in dilute-acid hydrolysis were identified as furan derivatives, e.g. furfural and 5-hydroxymethylfurfural
* Corresponding author. Tel.: /46-31-7723094; fax: /46-317723035 E-mail address: [email protected] (R. Millati).
(HMF), carboxylic acids, e.g. acetic acid, formic acid, and levulinic acid, and phenolic compounds [1]. Cultivation of the hydrolyzates can be carried out in different modes of operation including batch, fed-batch and continuous fermentation. The fermentation of hydrolyzates has traditionally been investigated in batch processes. Despite its simplicity, there is a major drawback of the batch fermentation, which is the high initial concentration of the inhibitors in the medium. It results in a long lag phase or sometimes even complete failure of the fermentation [2,3]. The fed-batch cultivation is of interest to overcome some of these problems [4,5]. This is due to the capability of the yeast for in situ detoxification of the hydrolyzates and conversion of the inhibitors such as furfural and HMF to some less toxic compounds [6 /8]. The capability of in situ detoxification of the cells makes continuous cultivation an attractive method for the fermentation of dilute-acid hydrolyzates. However, the dilution rate in continuous cultivation would be a function of the inhibitor concentrations, i.e. a lower
0032-9592/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 2 - 9 5 9 2 ( 0 2 ) 0 0 1 7 6 - 0
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dilution rate should be applied for a feed with higher concentration of inhibitors [9]. Furthermore, if a very low dilution rate is applied to ferment severely inhibiting hydrolyzates, the conversion rate of the inhibitors decreases with the decreased growth of the biomass [7,8]. This results in wash out of the cells from the bioreactor, even at very low dilution rates. Washout can be avoided, if the biomass is retained by e.g. filtration, recirculation or immobilization of the biomass. In a previous study [10], the continuous cultivation by immobilized Saccharomyces cerevisiae was shown to be able to ferment dilute-acid hydrolyzates with a high ethanol production rate. However, when the cells face a severely inhibiting hydrolyzate, metabolic activity and ethanol productivity decreased to zero, in spite of the presence of high cell mass concentration in the beads. Detoxification prior to fermentation is probably necessary to retain metabolically ‘active’ biomass, when a toxic hydrolyzate is used as feed. There are several detoxification methods including biological treatments, e.g. laccase and Trichoderma reesei , physical treatments, e.g. extraction, and chemical treatments, e.g. ion exchange, evaporation, overliming and sulphite treatment [11 /18]. Because of its low costs and high efficiency among the detoxification methods, overliming has been recognized as the most common treatment, although it has a drawback in terms of sugar losses [15]. Overliming is traditionally performed by the addition of alkali (Ca(OH)2 or NaOH) to increase the pH up to 10, followed by an adjustment to the cultivation pH [11,18]. The aim of the current work was to investigate the capability of overliming in detoxification and in increasing the fermentability of dilute-acid hydrolyzates in batch and continuous modes of operation. The effects of three variables in overliming were examined: pH, duration and temperature of detoxification. The fermentability of the detoxified hydrolyzates was examined by batch cultivation in shake flasks, and a selected hydrolyzate was further tested by continuous cultivation of immobilized S. cerevisiae in a bioreactor.
The pH of the hydrolyzates was less than 2. Leaving the hydrolyzate at room temperature for a week resulted in an average of 10% decrease in sugar concentration. 2.2. Overliming treatment The detoxification by the overliming procedure involved increasing the pH of the hydrolyzates to 10.0, 11.0 or 12.0 by addition of Ca(OH)2. The pH was held at the increased level for different periods of time 0, 1, 20 and 170 h, at two different temperatures, 25 and 60 8C. The pH of the detoxified hydrolyzates was then adjusted to the cultivation pH, followed by sterilization with autoclave, and the media were inoculated immediately. 2.3. Yeast strain and medium The yeast S. cerevisiae CBS 8066, obtained from the Central bureau voor Schimmel cultures (Delft, The Netherlands) was used in all experiments. The strain was maintained on agar plates made from yeast extract 10 g/l, soy peptone 20 g/l, and agar 20 g/l with D-glucose 20 g/l as an additional carbon source. Inoculum cultures were grown in 300-ml cotton-plugged-conical flasks on a shaker at 30 8C for 24 h. The liquid volume was 100 ml. The growth medium was a defined synthetic medium including 50 g/l glucose as previously reported [19]. 2.4. Immobilization procedure A 100-ml inoculum culture was mixed with 400 ml sterilized sodium alginate to give a final concentration of alginate solution of 3% (w/v). The beads were formed by slowly dropping the alginate solution into 1 l of gently stirred calcium chloride solution (30 g/l). The beads diameter formed through a small opening tube were between 3 and 4 mm [10]. The complete hardening of beads was achieved overnight. 2.5. Cultivation in shake flasks
2. Material and methods 2.1. Dilute-acid hydrolyzates The hydrolyzate used in the experiments was produced from forest residuals originated mainly from spruce [10]. The chips were hydrolyzed by 0.5% H2SO4 at 12/21 bar in a 350-l rebuilt masonite gun batch reactor in two stages. This has been described previously [10]. The hydrolyzates of the two stages were then mixed and stored at 4 8C before use. The hydrolyzate contained 20.0 g/l glucose, 18.1 g/l mannose, 4.2 g/l galactose, 7.8 g/l xylose, 1.95 g/l HMF, 0.8 g/l furfural, 3.0 g/l acetic acid and 2.4 g/l total phenolic compounds.
Cultivation in shake flasks was performed to test the fermentability of the detoxified hydrolyzates by overliming at different conditions. The detoxified hydrolyzates were pH-readjusted to 5.5 and then added to the culture medium, which was a defined synthetic medium with no carbon source similar to the inoculum. The cultivations were carried out in 300-ml cotton-pluggedErlenmeyer flasks placed in a shaker bath at a temperature of 30 8C for 48 h without pH controlling. The total liquid volume was 155 ml including 135-ml hydrolyzates, 5 ml inoculum, and 15 ml of a solution containing the other minerals and vitamins. Liquid samples were withdrawn at 0, 2, 5, 8, 24 and 48 h, which were analyzed by HPLC. Similar experiments were also
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carried out with undetoxified hydrolyzates. All the experiments were duplicated. 2.6. Continuous cultivation with immobilized cells Continuous cultivations were carried out in a BiostatA bioreactor (B. Braun Biotech International, Germany) at a temperature of 30 8C and stirring rate of 500 rpm with a working volume of 1.0 l. A detoxified hydrolyzate was fed into the bioreactor with a peristaltic pump (Watson-Marlow Alitea AB, Sweden) without pH re-adjustment. The pH value in the medium was controlled at 5.0 (9/0.07) by addition of 3 M HCl. Anaerobic conditions were maintained by continuous nitrogen sparging at a flow rate of 0.8 l/min controlled by a Hi-Tech mass flow controller (Ruurlo, The Netherlands). The cultivation was started in a batch mode in a fully defined medium [19] with glucose concentration of 50 g/l to prepare a high concentration of cells inside the beads. The hydrolyzates and the entire mineral and vitamins were then fed separately to the bioreactor at certain dilution rates. 2.7. Analytical methods The carbon dioxide and oxygen content in the outlet gas of the bioreactor were continuously measured with an acoustic gas monitor (model 1311, Innova, Denmark). Samples for HPLC analysis were withdrawn from the shake flasks or bioreactor, centrifuged and stored at /20 8C before analyzing. Glucose, xylose, galactose and mannose were analyzed on an Aminex HPX-87P column (Bio-Rad, USA) at 85 8C. Ultra-pure water was used as eluent at a flow rate of 0.6 ml/min. Ethanol, acetic acid, lactic acid, glycerol, furfural and HMF concentrations were determined by an Aminex HPX-87H column (Bio-Rad) at 60 8C with 0.6 ml/min eluent of 5 mM H2SO4. A refractive index (RI) detector (Waters 410, Millipore, Milford, USA) and an UV absorbance detector (Waters 486) were used in series. Concentrations of all mentioned metabolites were determined from the RI chromatograms, except lactic acid, furfural and HMF, which were determined from the UV chromatograms at 210 nm. The standard deviation of analyses by the HPLC was 3%. Total phenols concentration was analyzed by a spectrophotometric method with Folin & Ciocalteaus reagent at absorbance 725 nm [20].
3. Results 3.1. Detoxification The effect of three different parameters in detoxification by overliming with Ca(OH)2 was investigated:
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detoxification pH (pHd), time for detoxification (td) and temperature (Td). The detoxification was carried out by adjusting the temperature (Td), increasing the pH from the initial pH (i.e. less than 2) with calcium hydroxide to pHd, holding the pHd and Td unchanged for a period of time (td) and then decreasing the pH to the cultivation pH. These three variables were given as the following values: pHd f10; 11; 12g td f0; 1; 20; 170g (h) Td f25; 60g ( C) The concentration profiles of the sugars including glucose, mannose, galactose and xylose as well as furans (furfural and HMF), total phenolic compounds and acetic acid at different pHd, td and Td are summarized in Table 1 and Table 2. The concentrations of furans were highly affected by both the pHd and td, whereas there was not a clear difference between the furans’ profiles obtained at two different Td. The concentration of both furfural and HMF approached zero, while the pHd increased to 12 at the duration td higher than 20 h. However, the concentration of acetic acid was not generally affected by changing in any of the variables pHd, td or Td. The concentration of total phenolic compounds decreased by less than 30% at different pHd and td at Td /25 8C (Table 1). However, an increase in the phenolic compounds was observed at higher temperature and pHd (Table 2). The results show a general trend of decreasing sugar concentration, while pHd or td is increased. The maximum decrease in glucose concentration was more than 60%, when the hydrolyzate was treated at pHd 12 for td 170 h at 25 8C (Table 1). There was a similar trend for the other sugars, where the concentration of the total sugars was decreased by 68% at these treatment conditions. The missing sugars were generally found in the form of lactic acid (Fig. 1). 3.2. Batch cultivation of detoxified hydrolyzates Batch cultivations of the detoxified hydrolyzates were carried out in shake flasks, in order to examine the performance of the detoxification procedure. A portion of 135 ml of hydrolyzates was added to a total volume of 155 ml of fully synthetic medium containing 0.025 g of dry-weight yeast cells, and cultivated for 48 h. A summary of the most important results is presented in Fig. 2 and Fig. 3. None of the detoxified hydrolyzates at pHd 10 could be fermented, regardless of the treatment duration (td) and temperature (Td). In addition, no consumption of glucose and formation of ethanol were observed during the cultivation of the detoxified hydrolyzates at td /0. However, increasing either of these two variables, i.e.
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Table 1 The composition of hydrolyzates after different treatments of overliming at 25 8C Treatment pH (pHd)
Glucose (g/l)
Mannose (g/l)
Galactose (g/l)
Xylose (g/l)
HMF (g/l)
Furfural (g/l)
Acetic (g/l)
Total phenol (g/l)
0 0 0
10 11 12
20.1 20.1 18.6
18.4 20.1 16.6
4.4 3.5 3.6
7.8 7.7 7
2.03 0.78 1.51
0.40 0.82 0.28
2.8 3.0 2.4
2.5 2.4 2.4
1 1 1
10 11 12
19.8 19.4 17.4
17.9 18.1 15.8
4.1 2.8 3
7.5 7.3 6.4
1.91 1.32 0.49
0.37 0.64 0.06
2.8 2.8 2.5
2.4 2.4 2.2
20 20 20
10 11 12
20.6 19.6 13.5
19.7 20 12.7
3.7 3.3 0.6
7.3 6.6 2
1.43 0.56 0.02
0.27 0.06 0.00
2.7 2.7 2.7
2.3 2.2 2.1
170 170 170
10 11 12
18.5 18.9 10.6
16.5 18.7 9.5
3.1 3.4 1.3
7.5 7.2 1.8
1.19 0.35 0.04
0.29 0.06 0.00
2.5 2.9 2.5
2.4 1.8 2.7
Table 2 The composition of hydrolyzates after different treatments of overliming at 60 8C Treatment duration td (h)
Treatment pH (pHd)
Glucose (g/l)
Mannose (g/l)
Galactose (g/l)
Xylose (g/l)
HMF (g/l)
Furfural (g/l)
Acetic (g/l)
Total phenol (g/l)
0 0 0
10 11 12
19.5 19.4 19.8
17.6 18.4 19.8
3.9 3.2 3.4
7.6 7.3 7.3
1.96 1.89 1.59
0.38 0.89 0.74
2.7 3.1 3.1
2.3 1.7 2.3
1 1 1
10 11 12
19.1 20.0 18.4
17.3 20.1 19.8
3.8 3.5 2.4
7.5 7.4 6.1
1.57 1.39 0.35
0.28 0.63 0.17
2.3 3.0 3.1
2.4 1.8 6.0
20 20 20
10 11 12
20.4 16.1 5.8
20.4 16.9 5.7
3.6 2.9 1.2
7.3 5.4 0.9
1.55 0.08 0.02
0.28 0.02 0.00
2.8 2.8 2.9
2.3 3.0 4.5
170 170 170
10 11 12
18.7 15.5 7.1
17.1 14.2 6.6
3.1 2.7 1.3
7.3 5.7 1.0
1.35 0.27 0.04
0.35 0.05 0.00
2.8 2.7 2.9
2.1 2.6 3.6
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Treatment duration td (h)
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Fig. 1. The concentration profile of the fermentable sugars at different detoxification pHd of 10 (j), 11 (k) and 12 (') and produced lactic acid at pHd of 10 (I), 11 (m) and 12 (^) with various treatment durations (td) at two different Td of (a) 25 8C and (b) 60 8C.
pHd and td, will increase the fermentability. Among the 1 h detoxified hydrolyzates, the only ones with pHd 12 were fermentable, but with a long lag phase (Figs. 2 and 3). The lag phase of the hydrolyzate treated at 25 8C
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was somewhat shorter, since 1.5 g/l ethanol has been produced after 24 h cultivation. The fermentability of the treated hydrolyzates at pHd 11 was dependent on treatment duration, and increased by increasing ‘td’. While the hydrolyzates treated at this pHd for 0 and 1 h were not fermentable at either Td, treatment duration of 20 h at 25 8C made it fermentable but with a long lag phase (Fig. 2). Increasing the td to 170 h, cause the lag phase to be decreased. The treatment at higher temperature (Td /60 8C) resulted in a worse fermentability: at pHd 11 and td of 20 h the hydrolyzates were not fermentable, and by td of 170 h they were fermentable but with a long lag phase. The best fermentability was obtained at pHd 12, although td played an important role in the results. The quickly treated hydrolyzates at pHd 12, resulted in no fermentation within 48 h. However, when td increased to 1 h at either temperature of 25 or 60 8C, the detoxified hydrolyzates were fermentable but with a long lag phase (Figs. 2 and 3). Further increase in td to 20 or 170 h resulted in fully fermentable hydrolyzates. On the other hand, the increased td resulted in decline in the sugar concentration and formation of lactic acid (Fig. 1).
Fig. 2. The profile of ethanol production, glucose consumption and total furans for detoxified hydrolyzates at pHd of 10 (j), 11 (k) and 12 (') and various td of 0, 1, 20 and 170 h at 25 8C.
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Fig. 3. The profile of ethanol production, glucose consumption and total furans for detoxified hydrolyzates at pHd of 10 (j), 11 (k) and 12 (') and various td of 0, 1, 20 and 170 h at 60 8C.
3.3. Continuous cultivation of detoxified hydrolyzates The performance of detoxification was investigated in anaerobic continuous cultivation by immobilized cells of S cerevisiae. The inoculation medium (100 ml) containing 0.5 g dry cells was immobilized in a total volume of 500 ml beads of 3 /4 mm in diameter. The fermentation was started with a batch cultivation at pH 5 in a fully synthetic medium and 50 g/l glucose as carbon and energy source. The cells were grown up in the beads to reach a concentration of approximately 5 g/l. In order to check the fermentability of undetoxified hydrolyzates in continuous cultivation, it was fed to the
Fig. 4. Carbon dioxide evolution rate in continuous cultivation of the detoxified hydrolyzate by immobilized S. cerevisiae at two different dilution rates. The dashed lines show the points where the feed was changed.
bioreactor at a dilution rate (D ) of 0.1 and 0.2 h1 in different experiments. The feeding continued for 40 h, but the cells could not survive in either cases, carbon dioxide evolution rate (CER) declined to zero, and the residual glucose was increased to the level of glucose present in the feed. The continuous cultivation of a detoxified hydrolyzate was carried out similarly to the undetoxified one by addition of the hydrolyzate at dilution rates 0.2, 0.4, 0.6, 0.8 and 1.0 h 1. The selected hydrolyzate was overlimed at pHd 12, td of 20 h, and at Td of 25 8C. The treatment at these conditions was chosen due to its proper fermentability results in the batch cultivation. The cultivations were carried out and the CER was stabilized in all the stages. The cultivation continued at each stage for 5 residence times in order to be sure of achieving steady-state conditions. The feeding stopped between two stages until CER decreased to less than 2% of the CER at the steady-state conditions (e.g. cf. Fig. 4). A summary of the results at each dilution rate is presented in Table 3. The detoxification of the selected hydrolyzate was fully successful, since more than 80% of glucose and 70% of mannose were consumed even at high dilution rates. The residual glucose was zero at the lowest dilution rate and increased to 2.1 g/l at the highest D , i.e. 1.0 h1. The residual furfural and HMF were detected as less than 0.03 g/l in the bioreactors in
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Table 3 The residual sugars, furans and acetic acid in the medium during continuous cultivation of the detoxified hydrolyzate at pHd 12, td 20 h and Td 25 8C by immobilized S. cerevisiae at different dilution rates Concentration (g/l)
D 0.2 h 1
D 0.4 h 1
D 0.6 h 1
D 0.8 h 1
D 1.0 h 1
Residual glucosea Residual mannoseb Residual furfural Residual HMF Acetic acid
0 0.93 0.002 0.008 1.40
0.28 1.49 0.025 0.013 2.31
1.61 3.33 0.025 0.011 2.28
1.69 2.51 0.015 0.016 1.77
2.10 2.19 0.012 0.016 1.52
a b
Glucose concentration in the feed 12.1 g/l. Mannose concentration in the feed 11.4 g/l.
all the experiments. The concentration of acetic acid in the bioreactor was less than the corresponding concentration in the feed (Table 3), which means that part of the acid has been consumed by the yeast.
4. Discussion Detoxification by overliming is a promising method to increase the fermentability of the dilute-acid hydrolyzates. A conventional overliming is usually performed by adding of a base, e.g. Ca(OH)2 up to pH 10 or 11 at 25 or 60 8C, waiting for 30 /60 min and then decreasing the pH to a level suitable for the fermentation [11,15,16,18]. The results of the current work show the importance of choosing the right detoxification pH (pHd), duration (td) and temperature (Td). These variables can seriously affect the results of the detoxification process and the amount of sugars in the hydrolyzates. In general, the effect of these three variables on detoxification can be summarized as: 1) Any increase in pHd will enhance the detoxification and increase the fermentability of the hydrolyzates, but causes a decline in sugar concentration as well. 2) The detoxification process is not a quick process. The treatment duration (td) plays an important role in it, and longer duration increases the fermentability of the hydrolyzates. 3) The effect of temperature Td is not as drastic as the effects of pHd and td. The treatment at lower temperature generally resulted in a better fermentability. The effect of overliming is obvious in decreasing the concentration of furans (Tables 1 and 2), whereas the concentration of acetic acid remains unchanged and the total phenolic compounds decreases just a few percent. Among the phenolic compounds, less heavily substituted phenolics are probably the inhibitoriest materials in the hydrolyzates [21]. Phenol and vanillin are of the strongest phenolic compounds, but they have marginal inhibition effects at concentration of 1 g/l [22]. Although furans are not considered as strong inhibitors in such
low concentrations as appeared in the hydrolyzates [7 / 9], but it seems that the partial removal of furfural and HMF is enough to make the hydrolyzate fermentable. Acetic acid is sometimes considered as inhibitor and sometimes as enhancer for the fermentation [19,23]. Acetic acid would inhibit the fermentation, if the concentration of undissociated acetic acid increases to higher than 5 g/l. It is far from the condition present in the current work. The results of the current work are in the line of previous reports on the effects of overliming [11,16]. However, it should be noticed that the quantitative extrapolation of the values presented in the current or previous works to any other hydrolyzates should be carried out very carefully, since the hydrolysis process is not usually well reproducible. Achieving a better fermentability of the toxic hydrolyzates by overliming is at the cost of a decline in sugar concentration. The sugar degradation has not been so critical up to pHd 11 in a short time, whereas a major part of the sugar can be degraded at higher pHd in a longer td. The degradation of sugars was accompanied by appearance of lactic acid (Fig. 3), which is a known product of sugar decomposition in alkali solution [24,25]. The hexoses are believed to decompose first to 3-desoxy-hexosone, then to glyceraldehydes and methylglyoxal or metasaccharic acid, and then to lactic acid and glyceric aldehyde [24]. It can be concluded that overliming is not a simple process with defined variables. All the studied variables including detoxification pH, duration and temperature, and some other variables such as the type of base used, can affect the degree of detoxification. Since higher pHd or td gives better detoxification but at the cost of sugar depletion, an optimization for each batch of hydrolyzate is perhaps necessary in order to find the best overliming conditions.
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