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Yeast extract promotes phase shift of bio-butanol fermentation by Clostridium acetobutylicum ATCC824 using cassava as substrate
Bioresource Technology 125 (2012) 43–51
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Yeast extract promotes phase shift of bio-butanol fermentation by Clostridium acetobutylicum ATCC824 using cassava as substrate Xin Li ⇑, Zhigang Li, Junping Zheng, Zhongping Shi, Le Li The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China
h i g h l i g h t s " A severe delay of phase shift was found in fermentation using cassava substrate. " Yeast extract added during delay period promoted initiation of solventogenesis. " Performances of traditional and extractive runs were improved by yeast extract. " Real-time PCR was investigated to analyze transcriptional levels of genes. " This process can produce butanol from cassava, a cheap non-grain crop.
a r t i c l e
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Article history: Received 17 June 2012 Received in revised form 11 August 2012 Accepted 14 August 2012 Available online 24 August 2012 Keywords: Acetone–butanol–ethanol fermentation Phase shift Yeast extract Cassava Clostridium acetobutylicum ATCC824
a b s t r a c t When fermenting on cassava (15–25%, w/v) with Clostridium acetobutylicum ATCC824, a severe delay (18–40 h) was observed in the phase shift from acidogenesis to solventogenesis, compared to the cases of fermenting on corn. By adding yeast extract (2.5 g/L-broth) into cassava meal medium when the delay appeared, the phase shift was triggered and fermentation performances were consequently improved. Total butanol concentrations/butanol productivities, compared to those with cassava substrate alone, increased 15%/80% in traditional fermentation while 86%/79% in extractive fermentation using oleyl alcohol as the extractant, and reached the equivalent levels of those using corn substrate. Analysis of genetic transcriptional levels and measurements of free amino acids in the broth demonstrated that timely and adequate addition of yeast extract could promote phase shift by increasing transcriptional level of ctfAB to 16-fold, and indirectly enhance butanol synthesis through accelerating the accumulation of histidine and aspartic acid families. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Biofuel is one of the attractive means to prevent a further increase of carbon dioxide emissions. Currently, gasoline is blended with ethanol at various percentages (Durre, 2007). However, compared to bio-ethanol, bio-butanol has several advantages such as being less hygroscopic, less volatile, and miscible with gasoline/ diesel (Qureshi and Blaschek, 2001b; Qureshi and Ezeji, 2008). Renewable butanol is produced from the fermentations of carbohydrates in a process often referred as ABE fermentation, for solvent products of acetone, butanol and ethanol are formed at a ratio of 3:6:1 (w/w). ABE fermentation is a proven industrial process that uses solventogenic clostridia to convert starches or sugars ⇑ Corresponding author. Address: School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, Jiangsu Province, China. Tel./fax: +86 0510 85916276. E-mail addresses: [email protected] (X. Li), [email protected] (Z. Li), [email protected] (J. Zheng), [email protected] (Z. Shi), fireflyateens [email protected] (L. Li). 0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2012.08.056
into solvents (Green, 2011). However, the relatively high cost of conventional starch (corn) or sugar (molasses) substrates at arable land competing with foods production and the very low total solvents concentration (2%) have been identified as a major factor affecting the economic viability of ABE fermentation (Jones and Woods, 1986; Lee et al., 2008; Qureshi and Blaschek, 2001a; Qureshi et al., 2001; Wang and Blaschek, 2011). Some researchers have also attempted to use waste agricultural residues as the substrates, but cells were unable to grow on such feedstocks unless they were subject to a complex enzymatic hydrolysis pre-treatment (Qureshi et al., 2008, 2010a,b; Thirmal and Dahman, 2012). Among the available starch substances for bio-butanol production, cassava is very attractive because of being inexpensive, highly productive, and particularly non-competitive with foods for arable land (Cock, 1982). Although the utilization of cassava as a source of starch for production of butanol seems promising, recent reports indicated that butanol productivity when fermenting on cassava without any supplement is much lower than that of using corn starch or glucose (Gu et al., 2009; Lu et al., 2012; Thang et al., 2010).
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X. Li et al. / Bioresource Technology 125 (2012) 43–51
A major problem in ABE fermentation is the severe growth inhibition from butanol (Taya et al., 1985). To overcome this problem, in situ extractive fermentation was developed by removing butanol inhibitory effect, thus enhancing the fermentation productivity (Bankar et al., 2012; Dhamole et al., 2012; Roffler et al., 1987). Among various in situ fermentation extractants for butanol, oleyl alcohol has been recognized as one of the best because of being relatively non-toxic and having a high butanol distribution coefficient (Evans and Wang, 1988). Therefore, it is reasonable to use in situ extractive fermentation with oleyl alcohol as the extractant for investigating various behaviours/characteristics of ABE fermentation. In addition, butanol extractive fermentation using biodiesel as the extractant was also evaluated in our previous studies (Li et al., 2011; Zhang et al., 2008). Biodiesels have many advantages over the traditional fossil diesels particularly for its renewable and lower emission (lower exhaustion of SOx and NOx) features (Khan et al., 2009). However, in general, the lower cetane number (CN) and combustion power, as well as the ignition delay of biodiesels limit its practical application and promotion. The CN inspection report shown in our previous study (Zhang et al., 2008) indicated that CN value of the biodiesel extracting more than 10.0 g/L bio-butanol could increase by three points from 51.4 to 54.4, and the enhanced CN value was above the acceptable level for engine combustion. Therefore, biodiesel extracting certain amount butanol could be regarded as a properties improved fuel and used directly, while the intensive energy requirements for solvents products purification could be greatly relieved or partially eliminated. As a result, butanol productivity enhancement in ABE extractive fermentation with biodiesel as the extractant is closely associated with the performance improvement of the properties improved biodiesel production and the entire ABE fermentation processes. In this study, butanol fermentation by Clostridium acetobutylicum ATCC824 was conducted in a 7 L static and anaerobic fermentor. The objective of this work was to investigate the defects in ABE fermentation on cassava substrate and to explore the approaches to effectively enhance ABE fermentation performance using cassava as the substrate to replace the corn substrate, in both traditional and extractive fermentations. Fermentation performances of using cassava and corn as the substrates were compared with each other. 2. Methods 2.1. Microorganism C. acetobutylicum ATCC824 was maintained as spore suspension in 5% corn meal medium at 4 °C. The methods of inoculation and pre-culture followed those described in the literatures (Li et al., 2011; Zhang et al., 2008). 2.2. Substrates (media) preparation and in situ fermentative extractants pre-treatment The corn meal (raw starch content about 50% w/w) was obtained at local market and cassava meal (raw starch content about 65–70% w/w) was provided by Henan Tianguan Fuel Ethanol Co. Ltd., China. Both corn and cassava meals were sieved up to a mesh size 40. The medium was pretreated by adding a minuscule amount of a-amylase (8 U/g-corn or 8 U/g-cassava, heated in boiling water bath for 45 min) and then glucoamylase (120 U/g-corn or 120 U/g-cassava, heated at 62 °C for 60 min). Subsequently, the viscosity-reduced medium with natural pH was autoclaved at 121 °C for 15 min. a-amylase (20,000 U/mL) and glucoamylase (100,000 U/mL) were purchased from Genencor Biotech Co., Wuxi,
China. Oleyl alcohol (Tokyo Kasei Co. Ltd., Japan) and biodiesel (the chemically synthesized soybean one, provided by Huahong Biofuel Co., China) were used as the in situ extractants for the extractive fermentations. These extracants were either sterilized at 121 °C for 20 min or directly used without pre-treatment. Yeast extract was purchased from Angel Yeast Co. Ltd., Yichang, China. Concentrated yeast extract solution was sterilized at 115 °C for 30 min, and then pumped into the broth at certain fermentation instantly when gas production tended to cease and pH remained at low level (pH = 4.0–4.5) without rebound. After the addition, yeast extract concentration in broth reached a level of 2.5 g/L. 2.3. Fermentation method and condition Seed culture was carried out in 100 mL anaerobic fermentation bottles using corn meal as the substrate. The initial corn/cassava meal contents for traditional fermentations, extractive fermentations with biodiesel and oleyl alcohol as the extractants, were 15%, 15% and 25% (w/v), respectively. The fermentations were conducted in a 7 L static fermentor (Baoxing Bioengineering Co., China) equipped with pH and ORP (oxidative–reductive-potential) electrodes and a manual pressure adjustment unit. A temperature-controllable water bath (MP-10, Shanghai Permanent Science and Technology Co., China) was used to circulate hot water into the coil pipes settled inside the fermentor to maintain broth temperature at 37 °C. The fermentation medium loading volume ranged between 1.8–2.5 L, and equivalent volume of oleyl alcohol or biodiesel was added (for extractive fermentations) to ensure the volumetric ratio of oil/broth at 1:1. N2 was firstly sparged into the extractant reservoir to remove residual oxygen for 10 min. 10% (v/v) inoculum was transferred into the fermentor and the broth was also sparged with N2 for 10 min. In the extractive fermentations, the oxygen-free oleyl alcohol or biodiesel was poured into the fermentor using a peristaltic pump after inoculation. The initial pressure inside the fermentor was controlled at about 0.02 MPa (N2) to strictly maintain the anaerobic condition. The pressure gradually increased as fermentation started and self-generated gas began to evolve. The pressure was then controlled in a range of 0.030–0.055 MPa throughout fermentation. Agitation was occasionally done for a short time (5 min, 400 rpm) to promote butanol diffusion from aqueous phase into extractant phase. 2.4. Analytical methods The measurements of concentrations of solvents, organic acids and starch were the same as those described in our previous reports (Li et al., 2011; Zhang et al., 2008). After fermentations had proceeded for several hours, the total evolved gas amount was measured by collecting the gas in a graduated tube filled with water every hour. At 2–3 h intervals, the gas was firstly directed into two absorption bottles filled with 6 M NaOH solution and connected in-series to determine H2 production amount within a period of 15 min, and then the total evolved gas amount in the remaining 45 min (measurement window length of 1 h) was quantified without passing the gas through the absorption bottles. In this way, H2/CO2 ratio could be calculated using Eq. (1) with the assumption that H2 and CO2 were the only two components in the gas
H2 =CO2 ¼
AH2 4 AGAS 1:33 AH2 4
ð1Þ
where AH2 and AGAS were H2 formation amount measured in the first 15 min and total gas evolved amount in the remaining 45 min, respectively.
X. Li et al. / Bioresource Technology 125 (2012) 43–51
For measurements of free amino acids concentrations, the media were treated by 7% sulfosalicylic acid solution at volume ratio of 1:1, and for contents of amino acids released from proteolysis, corn and cassava meals were treated by 6 M HCl. The Agilent 1100 HPLC was used in the measurement, under the following conditions: reverse column ODS-C18 4.0 125 mm; temperature 40 °C; flow rate 1.0 mL/min; Eluent A 20 mmol/L Na-acetate; Eluent B 20 mmol/L of Na-acetate:methanol:acetonitrile = 1:2:2 (v/v); detection at Ex 340 nm and Em 450 nm with an fluorescence detector. 2.5. RNA purification, cDNA synthesis, and real-time fluorescence quantitative PCR analysis Total bacterial RNA was extracted using Trizol Plus RNA Purification Kit (Invitrogen™), and purification method described in the manual provided with the kit. Before starting RNA extraction, all the samples needed to be percolated through filter paper to remove cassava residues. Total RNA was used as the template to synthesize cDNA, and then cDNA products were amplified by the method of real-time fluorescence quantitative PCR with primers of ctfAB-F (50 -CAGAAAACGGAATAGTTGGAATG-30 ), ctfAB-R (50 TGACCACCACGGATTAGTGAA-30 ), adhE-F (50 -GTTTTGGCTATGTAT GAGGCTGA-30 ), adhE-R (50 -CAAGCGTGAAAGAAGGTGGTAT-30 ) bdhB-F (50 -ACGCTTCTGCCATTCTATCC-30 ) and bdhB-R (50 -ATT GCGGCACATCCAGATA-30 ). The following PCR conditions were adopted: an initial denaturation step at 95 °C for 10 min, followed by an amplification and quantification program repeating for 40 cycles (95 °C for 10 s, 60 °C for 60 s with a single fluorescence measurement), and a melting curve program (a continuous fluorescence measurement raising temperature from 60 to 95 °C with a slow heating unit). 3. Results and discussion 3.1. Comparison of butanol fermentation performance when using cassava and corn as the substrates In this study, C. acetobutylicum ATCC824 was cultivated on corn and cassava substrates to compare their fermentation performances. Both traditional and in situ extractive fermentations were conducted. The extractive fermentation (using oleyl alcohol as the extractant) was applied to investigate various features in ABE fermentations, since butanol inhibitory effect could be relieved or even eliminated, and fermentation could be extended for much longer time. Fig. 1A and B show the time courses of pH and gas production in traditional and extractive fermentations using corn (control) and cassava as feedstock. pH rebounded immediately after declining to their bottom levels when using corn substrate, for both traditional and extractive fermentations. In contrast, when using cassava substrate, pH dropped down to lower levels and then stayed at these levels without any changes for a long period. This period actually represented the time delay in phase shift, which is about 18 h for the traditional fermentation and 40 h for extractive one. In addition, total gas production when fermenting on the cassava substrate was much lower than that on corn substrate (control), especially for the extractive fermentation case. In the extractive fermentation cases, total gas production when using cassava substrate was 34 L/L-broth, which was only about 39% for the same procedures using corn substrate. As shown in Fig. 1C, final butanol concentration in the traditional fermentation using cassava substrate reached a relatively higher level of 11.85 g/L. However, butanol productivity in this case was much lower than that of fermenting on corn substrate,
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because the fermentation time was extended for about 20 h due to the time delay in phase shift. Furthermore, as shown in Fig. 1D, the final butanol concentrations in both aqueous phase (3.54 g/L) and extractant phase (15.5 g/L), in extractive fermentation using cassava as the substrate and oleyl alcohol as the extractant, were extremely lower than those of the extractive fermentation but using corn substrate (8.19 g/L in broth and 27.68 g/L in extractant phases). The fermentations using cassava substrate ended with very high residual starch concentrations, while the residual starch concentrations were much lower when using corn substrate (control), especially for the extractive fermentation. In addition, in both traditional and extractive fermentations on cassava substrate, the organic acids rise-up and drop-down patterns were quite different from those of fermentations on corn substrate (control): the acids accumulated slowly at early stage, and when the acids concentrations reached their peak values, they declined at a much slower rate than those obtained with corn substrate (control) (Fig. 1E and F). These results showed that in fermentation using cassava substrate, a severe time delay of phase shift occurred and the occurrence of phase shift delay significantly deteriorated fermentation performances, which could be reflected by the facts of very low rates of gas evolution, acids re-assimilation and solvent production. It should be addressed that in the fermentation operations using different substrates (cassava or corn), all of the operation conditions were the same except the sort of the substrates. On the other hand, the initial carbohydrate contents for traditional or extractive fermentations using different substrates were basically comparable, it was thus speculated that the nitrogen source including its type and contents deteriorated fermentation performance when using cassava substrate. As a result, the free amino acids contents in the two substrates (cassava and corn) after enzymatic pre-treatment (pre-treated medium) and complete hydrolysis were measured and then compared. Table 1 shows the measurement results of the total and individual free amino acids concentrations/contents in the two enzymatic pre-treated media and the two substrates after complete protein hydrolysis. The results indicated that the total free amino acids concentrations in two pre-treated media were basically comparable. However, the total amino acid content in cassava meal was only 1.81 g/100 g-cassava meal, which was much lower than that in corn meal (7.90 g/100 g-corn meal). These data suggested that protein content, namely the nitrogen source content, is severely insufficient in cassava meal. Thus, it could be speculated that the lack of nitrogen source in cassava meal may cause the phase shift delay, and therefore leading to the fermentation performance deterioration. On the other hand, pH dropped down to and then stayed at a very low level (4.0–4.5) without rebound for a long time in the fermentations using cassava substrate. Therefore, it was considered that the consecutively low level of pH might be another reason accounting for the fermentation performance deterioration.
3.2. Accelerating phase shift and improving fermentation performance by addition of yeast extract From the above results, it is speculated that there are two possible reasons accounting for the delay of phase shift triggering when using cassava substrate. One is the ‘‘acid crash’’ effect due to the severe acetate and butyrate accumulation but without any re-assimilation. It was reported that when an ‘‘acid crash’’ happens, the environment that is essential for shifting from acidogenic phase to solventogenic phase is deteriorated, so that the excessively produced acids cannot be converted into solvents normally (Maddox et al., 2000). Another reason is the lower contents of nitrogen sources and other necessary elements in the medium as pointed out previously.
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Fig. 1. Performance comparison of butanol fermentations using corn and cassava substrates. (A and B) pH and gas production. — / - - -: pH; ./5: gas production. (C and D) Butanol and residual starch concentrations. d/s: Butanol in aqueous phase; /e: butanol in extractant phase; N/4: residual starch. (E and F) Organic acids concentrations. j/ h: acetate; I/.: butyrate. (A and F) —: Cassava substrate; - - -: corn substrate. (A, C and E) Results of traditional butanol fermentation; (B, D and F) results of extractive butanol fermentation using oleyl alcohol as the extractant.
To deal with the potential ‘‘acid crash’’ problem, in ABE extractive fermentation using cassava substrate and with oleyl alcohol as the extractant, when pH continuously decreased without rebound and gas production tended to stop, ammonia water was added into broth to bring pH back to neutralized range (pH = 5.0–5.5) by force. However, after two attempts of adding ammonia water, as shown in Fig. 2A, pH continuously dropped down once again and gas production could not be recovered. As a result, it could be concluded that pH adjustment could not trigger the phase shift from acidogenic into solventogenic phase. Since regulating pH did not work, another rescue measure was taken by adding concentrated yeast extract solution (final concentration in medium was 2.5 g/L-broth) at 70 h of the fermentation. Yeast extract, enriched with nitrogen sources as proteins and amino acids, is one of the common used nitrogen sources for cell culture and fermentation processes. As shown in Fig. 2A, pH began to
rebound and gas production accelerated after addition of the concentrated yeast extract solution with only a 10 h response delay, and the phase shift occurred smoothly after that. As shown in Fig. 2B, final butanol concentrations in aqueous and extractant phases reached 8.04 and 27.34 g/L respectively, which were equivalent to those of using corn substrate. In addition, starch consumption was accelerated after adding the concentrated yeast extract solution, and starch was almost completely used out at the end of the fermentation. From the above results, it could be concluded that lower nitrogen contents in cassava substrate is the reason for the phase shift delay occurrence and the overall fermentation performance deterioration. To further verify the effectiveness of above mentioned manipulation and the relevant speculation, traditional and extractive fermentations (with oleyl alcohol as the extractant) using cassava substrate were conducted. Concentrated yeast extract solution
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X. Li et al. / Bioresource Technology 125 (2012) 43–51 Table 1 Amino acids concentrations in corn/cassava meal media and contents in corn/cassava meal after complete proteins hydrolysis. Medium
Concentrations of amino acids (g/L or g/100 g-meal) Total
His
Thr
Lys
Met
Tyr
Phe
Val
Leu
Corn
2.03E01a 7.90E+00b
4.27E03a 2.99E01b
1.94E05a 2.97E01b
2.77E03a 2.01E01b
5.67E02a 1.65E01b
5.57E03a 2.31E01b
5.51E03a 4.20E01b
4.40E04a 4.42E01b
3.30E03a 9.83E01b
Cassava
3.31E01a 1.81E+00b
6.24E03a 4.60E02 b
1.51E02a 6.84E02b
7.79E03a 6.38E02b
6.16E04a 4.95E02b
2.44E02a 4.53E02b
1.83E02a 7.59E02b
2.03E02a 1.16E01b
3.21E03a 1.23E01b
Cassava + yeast extractA
9.03E01a
1.67E02a
4.35E02a
4.65E02a
9.52E03a
6.60E02a
4.78E02a
7.11E02a
3.69E03a
His: histidine; Thr: threonine; Lys: lysine; Met: methionine; Tyr: tyrosine; Phe: phenylalanine; Val: valine; Leu: leucine. a Free amino acids concentrations in corn and cassava meal media after enzymatic pre-treatment (g/L). b Amino acids contents after complete proteins hydrolysis of corn or cassava meal (g/100 g-corn or cassava meal). A Concentration of yeast extract in the medium is 2.5 g/L-broth.
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Fig. 2. Fermentation course using cassava substrate subject to pH adjustment (at 34 and 56 h) and yeast extract addition (at 70 h). (A) pH and gas production. —: pH; .: gas production; B: butanol and residual starch concentrations. d: Butanol in aqueous phase; : butanol in extractant phase; N: residual starch.
(2.5 g/L-broth) was added during the phase shift delay period when gas production tended to cease. Fermentation performances were compared between the cases with and without yeast extract addition. As shown in Fig. 3A and B, after supplement of yeast extract for both traditional and extractive fermentations, the phase shift smoothly occurred about 10 h with pH rebounded whilst gas production recovered accordingly. Gas production, which is closely associated with the solvents production, increased significantly in fermentations with yeast extract addition as compared to those without. In clostridia metabolism, H2 and CO2 were the only two components in the evolved gas. H2 is generated in the electron transport
shuttle system by the catalysis of hydrogenase, and CO2 is generated in pyruvate ? acetyl-CoA and acetoacetyl-CoA ? acetone routes. When the acidogenic phase successfully shifts into the solventogenic phase, acetate and butyrate are re-assimilated and converted into acetyl-CoA and butyryl-CoA, respectively, accompanied with acetoacetyl-CoA converting into acetone. During this period, large amount of CO2 has to be released while H2 generation is decreased as butanol synthesis needs a large amount of NADH. Therefore, when the shift from acidogenic phase to solventogenic phase occurred, a reduced ratio of H2/CO2 could always be observed (Jones and Woods, 1986). As shown in Fig. 3A and B, when concentrated yeast extract solution was supplemented at 21 h during the traditional fermentation, H2/CO2 ratio was reduced rapidly from its top peak of 1.5 to a level lower than 0.25, and then lingered at low levels of 0.3–0.6 throughout the rest of fermentation period. For extractive fermentation case, H2/CO2 ratio also quickly decreased from 0.80 to lower levels of 0.25–0.55 after yeast extract addition at 39 h, and then stayed at these lower levels for about 25 h before going back to 0.80 again. In addition, the accumulated organic acids, acetate and butyrate, swiftly declined after yeast extract addition in both traditional and extractive fermentations, as shown in Fig. 3E and F. This suggested that acetate and butyrate are re-assimilated to form the precursors for butanol synthesis. Moreover, the two fermentation modes with the addition of concentrated yeast extract solution proceeded almost to completion whilst the concentrations of residual starch were reduced to lower level. As shown in Table 2 and Fig. 3C and D, When yeast extract was added in cassava meal medium during the delay period of phase shift in traditional fermentation, butanol productivity (0.27 g/L h1) was comparably higher than that of using corn substrate, and much higher (47–80%) than that of using cassava substrate but without yeast extract addition, as the phase shift delay was effectively shortened. At the same time, final butanol concentration (13.60 g/L) and butanol yield from starch (0.31 g/g-starch) were almost equivalent to those obtained with corn substrate. For the extractive fermentation with cassava substrate and yeast extract addition, total butanol concentration (34.37 g/L), butanol yield from starch (0.35 g/g-starch) and butanol productivity (0.34 g/L h1) were equivalent or even superior to those obtained with corn substrate. In particular, the selective butanol/acetone ratio, another very important performance index, strikingly reached 2.84, which is the highest among all fermentation operation modes. All these results pointed to a conclusion that timely and adequate addition of yeast extract could promote phase shift occurrence and improve fermentation performances comprehensively when using cassava substrate. The same operation procedure, timely and adequate addition of yeast extract, was also applied to the extractive fermentation with cassava substrate and using biodiesel as the extractant. In this case, butanol concentration in biodiesel, butanol yield from starch and
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Fig. 3. Performance comparison of butanol fermentation using cassava substrate with/without yeast extract addition during operation. (A and B) pH, H2/CO2 and gas production. — / - - -: pH; w: H2/CO2; ./5: gas production. (C and D) Butanol and residual starch concentrations. d/s: Butanol in aqueous phase; /e: butanol in extractant phase; N/4: residual starch. (E and F) Organic acids concentrations. j/h: Acetate; I/.: butyrate. (A–F): —: With yeast extract addition; - - -: without yeast extract addition. (A, C and E) Results of traditional butanol fermentation; (B, D and F) results of extractive butanol fermentation using oleyl alcohol as the extractant.
butanol productivity (10.56 g/L, 0.28 g/g-starch, 0.24 g/L h1) are all higher than those obtained with corn substrate (9.57 g/L, 0.25 g/g-starch, 0.20 g/L h1) and with cassava substrate but without yeast extract addition (8.16 g/L, 0.23 g/g-starch, 0.18 g/L h1). Butanol amount extracted in biodiesel, namely the butanol concentration in biodiesel, is a very important index reflecting the quality of the biodiesel. Higher butanol concentration in biodiesel directly leads to a higher biodiesel quality. In the case of using cassava substrate with yeast extract addition, butanol concentration in biodiesel reached a level of 10.56 g/L, which was 10% higher than that obtained with corn substrate, and this potentially contributed to the biodiesel quality improvement. The comparison results of fermentation performances under different conditions and operation modes were summarized in Table 2.
3.3. Effect of yeast extract supplement on genetic transcription level and accumulation of the amino acids stimulating butanol synthesis A typical feature of the clostridial solvent production is biphasic fermentation (Ezeji et al., 2007). The shift from acidogenic phase to solventogenic phase plays an important role during bio-butanol fermentation process, as the solvent products are formed smoothly only when the phase shift is triggered successfully. Currently, the factors triggering the metabolic shift are not clearly understood. Several investigations have reported that in batch cultures, the initiation of solvent production is associated with low extracellular and intracellular pH, as well as a high undissociated butyric acid concentration (Bahl et al., 1982; Monot et al., 1984). In this study, variations in genetic transcriptional levels of the key enzymes responsible for solvents production and major amino acids in broth
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X. Li et al. / Bioresource Technology 125 (2012) 43–51 Table 2 Fermentation performance under various operation modes with corn and cassava substrates. Batch #
Run time (h)
Final butanol in aqueous phase (g/L)
Total solvents (g/L) Butanol
Acetone
T1 T2 T3 T4 T5 T6 T7 E-OA1 E-OA2 E-OA3 E-OA4 E-OA5 E-OA6 E-BD1 E-BD2 E-BD3
65 68 80 51 60 38 22 102 100 92 162 101 99 90 83 78
11.63 13.74 11.80 13.60 12.95 4.41 1.27 8.19 6.21 3.54 8.04 7.07 6.60 8.80 6.57 7.81
11.63 13.74 11.80 13.61 12.95 4.41 1.27 35.87 27.15 19.04 35.38 34.37 29.81 18.37 (9.57a) 14.73 (8.16a) 18.37 (10.56a)
5.86 7.63 5.85 6.07 6.98 2.15 0.56 20.37 14.22 8.13 18.03 12.12 10.76 8.95 7.68 8.97
Butanol yield (g/gstarch)
Butanol productivity g/ (Lh)
Gas production amount (L/Lbroth)
0.25 0.27 0.27 0.31 0.28 0.10 0.08 0.23 0.26 0.26 0.22 0.35 0.29 0.25 0.23 0.28
0.18 0.20 0.15 0.27 0.22 0.12 0.06 0.35 0.27 0.19 0.22 0.34 0.30 0.20 0.18 0.24
23.26 37.36 17.72 39.71 29.85 11.62 7.67 86.13 78.96 34.35 81.56 77.74 60.09 32.87 26.36 42.66
T: Traditional fermentation runs; E-OA: Extractive fermentation runs with oleyl alcohol as the extractant; E-BD: Extractive fermentation runs with biodiesel as the extractant. T1-T2: corn substrate; T3: cassava substrate; T4–T5: cassava substrate and addition of yeast extract (2.5 g/L-broth) at 20 h and 16 h respectively; T6: corn substrate and addition of yeast extract (2.5 g/L-broth) at the beginning; T7: cassava substrate addition of yeast extract (2.5 g/L-broth) at the beginning. E-OA1–E-OA2: corn substrate; E-OA3–E-OA6: cassava substrate. E-OA3: normal run without yeast extract addition; E-OA4: adding ammonia water at 34 h and 55 h respectively and adding yeast extract at 71 h; E-OA5–E-OA6: adding yeast extract (2.5 g/L-broth) at 39 h. E-BD1: corn substrate; E-BD2: cassava substrate without yeast extract addition; E-BD3: cassava substrate and addition of yeast extract (2.5 g/L-broth) at 28 h. a Butanol concentration in bio-diesel.
during fermentations were analyzed or measured, attempting to explore the mechanism of how yeast extract addition promotes the phase shift and regulates butanol production. Nolling et al. (2001) proposed a metabolic map indicating the mechanism of butanol biosynthesis by C. acetobutylicum ATCC824. According to this map, the most important enzyme responsible for the phase shift from acidogenic to solventogenic phase is CoAtransferase which is encoded by ctfAB gene. CoA-transferase could re-assimilate the organic acids formed, and convert acetate and butyrate into acetyl-CoA and butyryl-CoA, the precursors of ethanol and butanol, respectively, whilst it could also convert acetoaceyl-CoA to acetoacetate, the precursor of acetone. On the other hand, butyraldehyde dehydrogenase (encoded by gene adhE) and butanol dehydrogenase (encoded by gene bdhB) are the two key enzymes directly associated with butanol biosynthesis. Here, the transcriptional levels of these genes during the extractive fermentations using oleyl alcohol as the extractant, with and without yeast extract supplement, were measured. 16S RNA in C. acetobutylicum ATCC 824 served as the internal reference. The gene transcriptional levels were quantified by PCR measurements. The normalized transcriptional levels (NTL) were then calculated based on Eq. (2) for the comparison and interpretation purposes, and the results were indicated in Fig. 4.
NTLjk ðiÞ ¼
TLjk ðiÞ Maxi;j fTLjk ðiÞg
i ¼ 1; N;
J ¼ 1; 2;
k ¼ 1; 3
ð2Þ
where TL is the measured (absolute) transcriptional levels, k represents the k-th gene (k = 1, 3; ctfAB, adhE or bdhB), j accounts for the comparison groups (j = 1, 2; with/without yeast extract addition), i refers to the ith measurement data (a total of N) within one comparison group and for one particular gene. As shown in Fig. 4A, the normalized transcriptional level of ctfAB, NTLctfAB, increased significantly and continuously from 0.063 to 1 with a maximal enhancement of 16-fold when yeast extract was supplemented at 39 h, while NTLctfAB without yeast extract addition remained at very low levels of 0.018–0.066 without any significant change. On the other hand, NTLadhE and
NTLbdhB, the normalized transcriptional levels of genes adhE and bdhB, varied irregularly for both cases (with and without yeast extract addition) during the fermentations, and no continuous and significant enhancements (>3-fold) were observed, as shown in Fig. 4B. These data indicated that the transcriptional level of ctfAB was considerably stimulated after yeast extract addition, and CoAtransferase was activated accordingly leading to smooth phase shift occurrence and acceleration of organic acids re-assimilation. CoA-transferase activation determined the butanol production enhancement after the yeast extract addition. Butyraldehyde dehydrogenase (encoded by adhE) and butanol dehydrogenase (encoded by bdhB) which are directly associated with butanol biosynthesis were not activated by yeast extract addition, thus they were not responsible for the butanol production enhancement. In cellular metabolism, amino acids are necessary for cell growth and also are important metabolic intermediates in ABE fermentation by C. acetobutylicum. It was reported that some amino acids, either self-generated or added, are favourable for butanol synthesis, but some others not. Masion et al. (1987) discovered that histidine has a beneficial effect on both solvents production and cell growth. Heluane et al. (2011) reported that lysine and methionine as butanol up-regulated products could enhance butanol production. Here in this study, we measured the major amino acids accumulated in cassava meal medium before and after yeast extract addition, to interpret the reason of butanol production enhancement after adding yeast extract from the other viewpoint. The results indicated that concentrations of histidine (histidine family), as well as threonine, lysine and methionine (aspartic acid family) in the broth increased remarkably during the period of 56– 66 h after yeast extract addition at 39 h, and then they decreased gradually (Fig. 5A). The maximal increments after yeast extract addition were 11-, 182-, 39- and 21-fold for histidine, threonine, lysine and methionine, respectively. Comparatively, when yeast extract was added into the fresh cassava meal medium, these amino acids only increased 3 (histidine), 3 (threonine), 6 (lysine) and 15-fold (methionine), as shown in Table 1. The huge increasing attitudes differences of these amino acids suggested that secre-
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X. Li et al. / Bioresource Technology 125 (2012) 43–51
A
A
0.10
0.003
NTLctfAB (-)
0.8 0.6 0.4 0.2 0.0
0.002 0.06
0.04 0.001 0.02
0.00 20
39 54 Fermentation time (h)
0.000 20
65
B 1.0 0.8 0.6 0.4
29 44 56 66 Fermentation time (h)
78
B
0.010 Concentrations of Tyr, Phe, Val and Leu (g/L)
NTLadhE and NTLbdhB (-)
0.08
Concentration of Met (g/L)
Concentrations of His, Thr and Lys (g/L)
1.0
0.008
0.006
0.004
0.002
0.2 0.000
0.0 20
39 54 Fermentation time (h)
20
65
Fig. 4. Changes in transcriptional levels of ctfAB, adhE and bdhB with or without yeast extract addition (at 39 h) in extractive fermentations using cassava substrate. (A) Transcriptional level of ctfAB. White bars: without yeast extract addition; black bars: with yeast extract addition. (B) Transcriptional levels of adhE and bdhB. White bars: adhE, without yeast extract addition; black bars: adhE, with yeast extract addition; slashed shadow bars: bdhB, without yeast extract addition; parallel shadow bars: bdhB, with yeast extract addition.
tions of histidine, threonine, lysine and methionine were not brought by the contents of yeast extract itself, but by cellular production simulated by yeast extract addition. Then the over-produced histidine and aspartic acid families enhanced butanol production in turn. On the other hand, as shown in Fig. 5B, amino acids from aromatic family (including tyrosine and phenylalanine) and pyruvate family (valine and leucine), which were not reported to have positive effects on butanol synthesis, did not change significantly after yeast extract addition in cassava meal medium. From the above mentioned results, it could be concluded that addition of yeast extract during ABE fermentations on cassava substrate activated CoA-transferase and stimulated the secretions of histidine and aspartic acid families, thus, butanol productions were largely enhanced. Then, there is an argument whether addition of yeast extract into cassava or corn substrate at the beginning of the fermentations might also enhance ABE fermentation performance? To clarify the doubt, concentrated yeast extract solution (2.5 g/L-broth) was added at the beginning of traditional fermentations on both corn and cassava substrates. The results showed that fermentations ceased at about 30 h without phase shift occurring for both cases. Final butanol concentrations were very low (4.41 g/L for corn substrate case and 1.27 g/L for cassava substrate case, Table 2). In contrast, concentrations of acetate and butyrate were high without reduction until the end of fermentation (2.56
29 44 56 66 Fermentation time (h)
78
Fig. 5. Changes of certain amino acids in cassava meal medium during extractive fermentation with yeast extract addition at 39 h. (A) The accumulations of histidine and aspartic acid families. Black bars: histidine (His); white bars: threonine (Thr); slashed shadow bars: lysine (Lys); parallel shadow bars: methionine (Met). (B) The accumulations of pyruvatic acid and aromatic acid families. Black bars: tyrosine (Tyr); white bars: phenylalanine (Phe); slashed shadow bars: valine (Val); parallel shadow bars: leucine (Leu).
and 1.34 g/L for corn substrate case, 2.34 and 0.62 g/L for cassava substrate case). With this operation mode, the normal metabolism on both corn and cassava substrates was destroyed and phase shift did not happen, suggesting that activation of CoA-transferase and over-secretions of amino acids would never occur in this case. These results demonstrated that yeast extract addition has the effects on promoting phase shift occurrence and enhancing fermentation performances using cassava substrate, when and only when it is added during the delay period. 4. Conclusions Cassava, a perspective substrate for bio-butanol production, was deficient in nitrogen source, leading to a severe phase shift delay and low butanol productivity. When yeast extract was added during the delay period of fermentation on cassava substrate, phase shift was triggered subsequently, and entire fermentation performances reached equivalent levels of those using corn substrate. Analysis of genetic transcriptional levels demonstrated that yeast extract could promote phase shift by largely increasing the transcription level of ctfAB to 16-fold. Moreover, yeast extract could stimulate secretions of amino acids from histidine and aspartic acid families, which could further enhance bio-butanol production.
X. Li et al. / Bioresource Technology 125 (2012) 43–51
Acknowledgements The study was supported by National Natural Science Foundation Program (#20976072) and Major State Basic Research Development Program (#2007CB714303) of China. The authors also appreciated assistance from Mr. Jiang Xu and Mr. Martin D’Ambrosio in refining English of the manuscript. References Bahl, H., Andersch, W., Braun, K., Gottschalk, G., 1982. Effect of pH and butyrate concentration on the production of acetone and butanol by Clostridium acetobutylicum grown in continuous culture. Appl. Microbiol. Biotechnol. 14, 17–20. Bankar, S.B., Survase, S.A., Singhal, R.S., Granstrom, T., 2012. Continuous two stage acetone–butanol–ethanol fermentation with integrated solvent removal using Clostridium acetobutylicum B 5313. Bioresour. Technol. 106, 110–116. Cock, J.H., 1982. Cassava: a basic energy source in the tropics. Science 218, 755–762. Dhamole, P.B., Wang, Z., Liu, Y., Wang, B., Feng, H., 2012. Extractive fermentation with non-ionic surfactants to enhance butanol production. Biomass Bioenerg. 40, 112–119. Durre, P., 2007. Biobutanol: an attractive biofuel. J. Biotechnol. 2, 1525–1534. Evans, P.J., Wang, H.Y., 1988. Enhancement of butanol formation by Clostridium acetobutylicum in the presence of decanol-oleyl alcohol mixed extractants. Appl. Environ. Microb. 54, 1662–1667. Ezeji, T.C., Qureshi, N., Blaschek, H.P., 2007. Bioproduction of butanol from biomass: from genes to bioreactors. Curr. Opin. Biotech. 18, 220–227. Green, E.M., 2011. Fermentative production of butanol-the industrial perspective. Curr. Opin. Biotech. 22, 337–343. Gu, Y., Hu, S., Chen, J., Shao, L., He, H., Yang, Y., Yang, S., Jiang, W., 2009. Ammonium acetate enhances solvent production by Clostridium acetobutylicum EA 2018 using cassava as a fermentation medium. J. Ind. Microbiol. Biotechnol. 36, 1225–1232. Heluane, H., Evans, M.R., Dagher, S.F., Bruno-Barcena, J.M., 2011. Meta-analysis and functional validation of nutritional requirements of solventogenic clostridia growing under butanol stress conditions and coutilization of D-glucose and Dxylose. Appl. Environ. Microb. 77, 4473–4485. Jones, D.T., Woods, D.R., 1986. Acetone–butanol fermentation revisited. Microbiol. Res. 50, 484–524. Khan, S.A., Rashmi, Hussain, M.Z., Prasad, S., Banerjee, U.C., 2009. Prospects of biodiesel production from microalgae in India. Renew. Sust. Energ. Rev. 13, 2361–2372. Lee, S.Y., Park, J.H., Jang, S.H., Nielsen, L.K., Kim, J., Jung, K.S., 2008. Fermentative butanol production by clostridia. Biotechnol. Bioeng. 101, 209–228. Li, Z., Li, X., Zheng, J., Zhang, S., Shi, Z., 2011. Butanol extractive fermentation to simultaneously produce ‘‘properties improved’’ biodiesel and butanol in a water and energy-saving operation way. J. Biobased Mater. Bioenergy 5, 312–318. Lu, C., Zhao, J., Yang, S.-T., Wei, D., 2012. Fed-batch fermentation for n-butanol production from cassava bagasse hydrolysate in a fibrous bed bioreactor with continuous gas stripping. Bioresour. Technol. 104, 380–387.
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Maddox, I.S., Steiner, E., Hirsch, S., Wessner, S., Gutierrez, N.A., Gapes, J.R., Schuster, K.C., 2000. The cause of ‘‘acid-crash’’ and ‘‘acidogenic fermentations’’ during the batch acetone–butanol–ethanol (ABE) fermentation process. J. Mol. Microb. Biotech. 2, 95–100. Masion, E., Amine, J., Marczak, R., 1987. Influence of amino acid supplements on the metabolism of Clostridium acetobutylicum. FEMS Microbiol. Lett. 43, 269–274. Monot, F., Engasser, J.M., Petitdemange, H., 1984. Influence of pH and undissociated butyric acid on the production of acetone and butanol in batch cultures of Clostridium acetobutylicum. Appli. Microbiol. Biotechnol. 19, 422–426. Nolling, J., Breton, G., Omelchenko, M.V., Makarova, K.S., Zeng, Q.D., Gibson, R., Lee, H.M., Dubois, J., Qiu, D.Y., Hitti, J., Wolf, Y.I., Tatusov, R.L., Sabathe, F., DoucetteStamm, L., Soucaille, P., Daly, M.J., Bennett, G.N., Koonin, E.V., Smith, D.R., Finishing, G.T.C.S.C.P., 2001. Genome sequence and comparative analysis of the solvent-producing bacterium Clostridium acetobutylicum. J. Bacteriol. 183, 4823–4838. Qureshi, N., Blaschek, H.P., 2001a. ABE production from corn: a recent economic evaluation. J. Ind. Microbiol. Biotechnol. 27, 292–297. Qureshi, N., Blaschek, H.P., 2001b. Evaluation of recent advances in butanol fermentation, upstream, and downstream processing. Bioproc. Biosyst. Eng. 24, 219–226. Qureshi, N., Ezeji, T.C., 2008. Butanol, ‘a superior biofuel’ production from agricultural residues (renewable biomass): recent progress in technology. Biofuel. Bioprod. Biorefin. 2, 319–330. Qureshi, N., Ezeji, T.C., Ebener, J., Dien, B.S., Cotta, M.A., Blaschek, H.P., 2008. Butanol production by Clostridium beijerinckii. Part I: use of acid and enzyme hydrolyzed corn fiber. Bioresour. Technol. 99, 5915–5922. Qureshi, N., Lolas, A., Biaschek, H.P., 2001. Soy molasses as fermentation substrate for production of butanol using Clostridium beijerinckii BA101. J. Ind. Microbiol. Biotechnol. 26, 290–295. Qureshi, N., Saha, B.C., Dien, B., Hector, R.E., Cotta, M.A., 2010a. Production of butanol (a biofuel) from agricultural residues. Part I – use of barley straw hydrolysate. Biomass Bioenerg. 34, 559–565. Qureshi, N., Saha, B.C., Hector, R.E., Dien, B., Hughes, S., Liu, S., Iten, L., Bowman, M.J., Sarath, G., Cotta, M.A., 2010b. Production of butanol (a biofuel) from agricultural residues: Part II – use of corn stover and switchgrass hydrolysates. Biomass Bioenerg. 34, 566–571. Roffler, S.R., Blanch, H.W., Wilke, C.R., 1987. In-situ recovery of butanol during fermentation. Bioproc. Biosyst. Eng. 2, 1–12. Taya, M., Ishii, S., Kobayashi, T., 1985. Monitoring and control for extractive fermentation of Clostridium acetobutylicum. J. Ferment. Technol. 63, 181–187. Thang, V.H., Kanda, K., Kobayashi, G., 2010. Production of acetone–butanol–ethanol (ABE) in direct fermentation of cassava by Clostridium saccharoperbutylacetonicum N1–4. Appl. Biochem. Biotech. 161, 157–170. Thirmal, C., Dahman, Y., 2012. Comparisons of existing pretreatment, saccharification, and fermentation processes for butanol production from agricultural residues. Can. J. Chem. Eng. 90, 745–761. Wang, Y., Blaschek, H.P., 2011. Optimization of butanol production from tropical maize stalk juice by fermentation with Clostridium beijerinckii NCIMB 8052. Bioresour. Technol. 102, 9985–9990. Zhang, L., Yang, Y., Shi, Z., 2008. Performance optimization of property-improved biodiesel manufacturing process coupled with butanol extractive fermentation. Chin. J. Biotechnol. 24, 1943–1948.
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Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Yeast extract promotes phase shift of bio-butanol fermentation by Clostridium acetobutylicum ATCC824 using cassava as substrate Xin Li ⇑, Zhigang Li, Junping Zheng, Zhongping Shi, Le Li The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China
h i g h l i g h t s " A severe delay of phase shift was found in fermentation using cassava substrate. " Yeast extract added during delay period promoted initiation of solventogenesis. " Performances of traditional and extractive runs were improved by yeast extract. " Real-time PCR was investigated to analyze transcriptional levels of genes. " This process can produce butanol from cassava, a cheap non-grain crop.
a r t i c l e
i n f o
Article history: Received 17 June 2012 Received in revised form 11 August 2012 Accepted 14 August 2012 Available online 24 August 2012 Keywords: Acetone–butanol–ethanol fermentation Phase shift Yeast extract Cassava Clostridium acetobutylicum ATCC824
a b s t r a c t When fermenting on cassava (15–25%, w/v) with Clostridium acetobutylicum ATCC824, a severe delay (18–40 h) was observed in the phase shift from acidogenesis to solventogenesis, compared to the cases of fermenting on corn. By adding yeast extract (2.5 g/L-broth) into cassava meal medium when the delay appeared, the phase shift was triggered and fermentation performances were consequently improved. Total butanol concentrations/butanol productivities, compared to those with cassava substrate alone, increased 15%/80% in traditional fermentation while 86%/79% in extractive fermentation using oleyl alcohol as the extractant, and reached the equivalent levels of those using corn substrate. Analysis of genetic transcriptional levels and measurements of free amino acids in the broth demonstrated that timely and adequate addition of yeast extract could promote phase shift by increasing transcriptional level of ctfAB to 16-fold, and indirectly enhance butanol synthesis through accelerating the accumulation of histidine and aspartic acid families. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Biofuel is one of the attractive means to prevent a further increase of carbon dioxide emissions. Currently, gasoline is blended with ethanol at various percentages (Durre, 2007). However, compared to bio-ethanol, bio-butanol has several advantages such as being less hygroscopic, less volatile, and miscible with gasoline/ diesel (Qureshi and Blaschek, 2001b; Qureshi and Ezeji, 2008). Renewable butanol is produced from the fermentations of carbohydrates in a process often referred as ABE fermentation, for solvent products of acetone, butanol and ethanol are formed at a ratio of 3:6:1 (w/w). ABE fermentation is a proven industrial process that uses solventogenic clostridia to convert starches or sugars ⇑ Corresponding author. Address: School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, Jiangsu Province, China. Tel./fax: +86 0510 85916276. E-mail addresses: [email protected] (X. Li), [email protected] (Z. Li), [email protected] (J. Zheng), [email protected] (Z. Shi), fireflyateens [email protected] (L. Li). 0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2012.08.056
into solvents (Green, 2011). However, the relatively high cost of conventional starch (corn) or sugar (molasses) substrates at arable land competing with foods production and the very low total solvents concentration (2%) have been identified as a major factor affecting the economic viability of ABE fermentation (Jones and Woods, 1986; Lee et al., 2008; Qureshi and Blaschek, 2001a; Qureshi et al., 2001; Wang and Blaschek, 2011). Some researchers have also attempted to use waste agricultural residues as the substrates, but cells were unable to grow on such feedstocks unless they were subject to a complex enzymatic hydrolysis pre-treatment (Qureshi et al., 2008, 2010a,b; Thirmal and Dahman, 2012). Among the available starch substances for bio-butanol production, cassava is very attractive because of being inexpensive, highly productive, and particularly non-competitive with foods for arable land (Cock, 1982). Although the utilization of cassava as a source of starch for production of butanol seems promising, recent reports indicated that butanol productivity when fermenting on cassava without any supplement is much lower than that of using corn starch or glucose (Gu et al., 2009; Lu et al., 2012; Thang et al., 2010).
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X. Li et al. / Bioresource Technology 125 (2012) 43–51
A major problem in ABE fermentation is the severe growth inhibition from butanol (Taya et al., 1985). To overcome this problem, in situ extractive fermentation was developed by removing butanol inhibitory effect, thus enhancing the fermentation productivity (Bankar et al., 2012; Dhamole et al., 2012; Roffler et al., 1987). Among various in situ fermentation extractants for butanol, oleyl alcohol has been recognized as one of the best because of being relatively non-toxic and having a high butanol distribution coefficient (Evans and Wang, 1988). Therefore, it is reasonable to use in situ extractive fermentation with oleyl alcohol as the extractant for investigating various behaviours/characteristics of ABE fermentation. In addition, butanol extractive fermentation using biodiesel as the extractant was also evaluated in our previous studies (Li et al., 2011; Zhang et al., 2008). Biodiesels have many advantages over the traditional fossil diesels particularly for its renewable and lower emission (lower exhaustion of SOx and NOx) features (Khan et al., 2009). However, in general, the lower cetane number (CN) and combustion power, as well as the ignition delay of biodiesels limit its practical application and promotion. The CN inspection report shown in our previous study (Zhang et al., 2008) indicated that CN value of the biodiesel extracting more than 10.0 g/L bio-butanol could increase by three points from 51.4 to 54.4, and the enhanced CN value was above the acceptable level for engine combustion. Therefore, biodiesel extracting certain amount butanol could be regarded as a properties improved fuel and used directly, while the intensive energy requirements for solvents products purification could be greatly relieved or partially eliminated. As a result, butanol productivity enhancement in ABE extractive fermentation with biodiesel as the extractant is closely associated with the performance improvement of the properties improved biodiesel production and the entire ABE fermentation processes. In this study, butanol fermentation by Clostridium acetobutylicum ATCC824 was conducted in a 7 L static and anaerobic fermentor. The objective of this work was to investigate the defects in ABE fermentation on cassava substrate and to explore the approaches to effectively enhance ABE fermentation performance using cassava as the substrate to replace the corn substrate, in both traditional and extractive fermentations. Fermentation performances of using cassava and corn as the substrates were compared with each other. 2. Methods 2.1. Microorganism C. acetobutylicum ATCC824 was maintained as spore suspension in 5% corn meal medium at 4 °C. The methods of inoculation and pre-culture followed those described in the literatures (Li et al., 2011; Zhang et al., 2008). 2.2. Substrates (media) preparation and in situ fermentative extractants pre-treatment The corn meal (raw starch content about 50% w/w) was obtained at local market and cassava meal (raw starch content about 65–70% w/w) was provided by Henan Tianguan Fuel Ethanol Co. Ltd., China. Both corn and cassava meals were sieved up to a mesh size 40. The medium was pretreated by adding a minuscule amount of a-amylase (8 U/g-corn or 8 U/g-cassava, heated in boiling water bath for 45 min) and then glucoamylase (120 U/g-corn or 120 U/g-cassava, heated at 62 °C for 60 min). Subsequently, the viscosity-reduced medium with natural pH was autoclaved at 121 °C for 15 min. a-amylase (20,000 U/mL) and glucoamylase (100,000 U/mL) were purchased from Genencor Biotech Co., Wuxi,
China. Oleyl alcohol (Tokyo Kasei Co. Ltd., Japan) and biodiesel (the chemically synthesized soybean one, provided by Huahong Biofuel Co., China) were used as the in situ extractants for the extractive fermentations. These extracants were either sterilized at 121 °C for 20 min or directly used without pre-treatment. Yeast extract was purchased from Angel Yeast Co. Ltd., Yichang, China. Concentrated yeast extract solution was sterilized at 115 °C for 30 min, and then pumped into the broth at certain fermentation instantly when gas production tended to cease and pH remained at low level (pH = 4.0–4.5) without rebound. After the addition, yeast extract concentration in broth reached a level of 2.5 g/L. 2.3. Fermentation method and condition Seed culture was carried out in 100 mL anaerobic fermentation bottles using corn meal as the substrate. The initial corn/cassava meal contents for traditional fermentations, extractive fermentations with biodiesel and oleyl alcohol as the extractants, were 15%, 15% and 25% (w/v), respectively. The fermentations were conducted in a 7 L static fermentor (Baoxing Bioengineering Co., China) equipped with pH and ORP (oxidative–reductive-potential) electrodes and a manual pressure adjustment unit. A temperature-controllable water bath (MP-10, Shanghai Permanent Science and Technology Co., China) was used to circulate hot water into the coil pipes settled inside the fermentor to maintain broth temperature at 37 °C. The fermentation medium loading volume ranged between 1.8–2.5 L, and equivalent volume of oleyl alcohol or biodiesel was added (for extractive fermentations) to ensure the volumetric ratio of oil/broth at 1:1. N2 was firstly sparged into the extractant reservoir to remove residual oxygen for 10 min. 10% (v/v) inoculum was transferred into the fermentor and the broth was also sparged with N2 for 10 min. In the extractive fermentations, the oxygen-free oleyl alcohol or biodiesel was poured into the fermentor using a peristaltic pump after inoculation. The initial pressure inside the fermentor was controlled at about 0.02 MPa (N2) to strictly maintain the anaerobic condition. The pressure gradually increased as fermentation started and self-generated gas began to evolve. The pressure was then controlled in a range of 0.030–0.055 MPa throughout fermentation. Agitation was occasionally done for a short time (5 min, 400 rpm) to promote butanol diffusion from aqueous phase into extractant phase. 2.4. Analytical methods The measurements of concentrations of solvents, organic acids and starch were the same as those described in our previous reports (Li et al., 2011; Zhang et al., 2008). After fermentations had proceeded for several hours, the total evolved gas amount was measured by collecting the gas in a graduated tube filled with water every hour. At 2–3 h intervals, the gas was firstly directed into two absorption bottles filled with 6 M NaOH solution and connected in-series to determine H2 production amount within a period of 15 min, and then the total evolved gas amount in the remaining 45 min (measurement window length of 1 h) was quantified without passing the gas through the absorption bottles. In this way, H2/CO2 ratio could be calculated using Eq. (1) with the assumption that H2 and CO2 were the only two components in the gas
H2 =CO2 ¼
AH2 4 AGAS 1:33 AH2 4
ð1Þ
where AH2 and AGAS were H2 formation amount measured in the first 15 min and total gas evolved amount in the remaining 45 min, respectively.
X. Li et al. / Bioresource Technology 125 (2012) 43–51
For measurements of free amino acids concentrations, the media were treated by 7% sulfosalicylic acid solution at volume ratio of 1:1, and for contents of amino acids released from proteolysis, corn and cassava meals were treated by 6 M HCl. The Agilent 1100 HPLC was used in the measurement, under the following conditions: reverse column ODS-C18 4.0 125 mm; temperature 40 °C; flow rate 1.0 mL/min; Eluent A 20 mmol/L Na-acetate; Eluent B 20 mmol/L of Na-acetate:methanol:acetonitrile = 1:2:2 (v/v); detection at Ex 340 nm and Em 450 nm with an fluorescence detector. 2.5. RNA purification, cDNA synthesis, and real-time fluorescence quantitative PCR analysis Total bacterial RNA was extracted using Trizol Plus RNA Purification Kit (Invitrogen™), and purification method described in the manual provided with the kit. Before starting RNA extraction, all the samples needed to be percolated through filter paper to remove cassava residues. Total RNA was used as the template to synthesize cDNA, and then cDNA products were amplified by the method of real-time fluorescence quantitative PCR with primers of ctfAB-F (50 -CAGAAAACGGAATAGTTGGAATG-30 ), ctfAB-R (50 TGACCACCACGGATTAGTGAA-30 ), adhE-F (50 -GTTTTGGCTATGTAT GAGGCTGA-30 ), adhE-R (50 -CAAGCGTGAAAGAAGGTGGTAT-30 ) bdhB-F (50 -ACGCTTCTGCCATTCTATCC-30 ) and bdhB-R (50 -ATT GCGGCACATCCAGATA-30 ). The following PCR conditions were adopted: an initial denaturation step at 95 °C for 10 min, followed by an amplification and quantification program repeating for 40 cycles (95 °C for 10 s, 60 °C for 60 s with a single fluorescence measurement), and a melting curve program (a continuous fluorescence measurement raising temperature from 60 to 95 °C with a slow heating unit). 3. Results and discussion 3.1. Comparison of butanol fermentation performance when using cassava and corn as the substrates In this study, C. acetobutylicum ATCC824 was cultivated on corn and cassava substrates to compare their fermentation performances. Both traditional and in situ extractive fermentations were conducted. The extractive fermentation (using oleyl alcohol as the extractant) was applied to investigate various features in ABE fermentations, since butanol inhibitory effect could be relieved or even eliminated, and fermentation could be extended for much longer time. Fig. 1A and B show the time courses of pH and gas production in traditional and extractive fermentations using corn (control) and cassava as feedstock. pH rebounded immediately after declining to their bottom levels when using corn substrate, for both traditional and extractive fermentations. In contrast, when using cassava substrate, pH dropped down to lower levels and then stayed at these levels without any changes for a long period. This period actually represented the time delay in phase shift, which is about 18 h for the traditional fermentation and 40 h for extractive one. In addition, total gas production when fermenting on the cassava substrate was much lower than that on corn substrate (control), especially for the extractive fermentation case. In the extractive fermentation cases, total gas production when using cassava substrate was 34 L/L-broth, which was only about 39% for the same procedures using corn substrate. As shown in Fig. 1C, final butanol concentration in the traditional fermentation using cassava substrate reached a relatively higher level of 11.85 g/L. However, butanol productivity in this case was much lower than that of fermenting on corn substrate,
45
because the fermentation time was extended for about 20 h due to the time delay in phase shift. Furthermore, as shown in Fig. 1D, the final butanol concentrations in both aqueous phase (3.54 g/L) and extractant phase (15.5 g/L), in extractive fermentation using cassava as the substrate and oleyl alcohol as the extractant, were extremely lower than those of the extractive fermentation but using corn substrate (8.19 g/L in broth and 27.68 g/L in extractant phases). The fermentations using cassava substrate ended with very high residual starch concentrations, while the residual starch concentrations were much lower when using corn substrate (control), especially for the extractive fermentation. In addition, in both traditional and extractive fermentations on cassava substrate, the organic acids rise-up and drop-down patterns were quite different from those of fermentations on corn substrate (control): the acids accumulated slowly at early stage, and when the acids concentrations reached their peak values, they declined at a much slower rate than those obtained with corn substrate (control) (Fig. 1E and F). These results showed that in fermentation using cassava substrate, a severe time delay of phase shift occurred and the occurrence of phase shift delay significantly deteriorated fermentation performances, which could be reflected by the facts of very low rates of gas evolution, acids re-assimilation and solvent production. It should be addressed that in the fermentation operations using different substrates (cassava or corn), all of the operation conditions were the same except the sort of the substrates. On the other hand, the initial carbohydrate contents for traditional or extractive fermentations using different substrates were basically comparable, it was thus speculated that the nitrogen source including its type and contents deteriorated fermentation performance when using cassava substrate. As a result, the free amino acids contents in the two substrates (cassava and corn) after enzymatic pre-treatment (pre-treated medium) and complete hydrolysis were measured and then compared. Table 1 shows the measurement results of the total and individual free amino acids concentrations/contents in the two enzymatic pre-treated media and the two substrates after complete protein hydrolysis. The results indicated that the total free amino acids concentrations in two pre-treated media were basically comparable. However, the total amino acid content in cassava meal was only 1.81 g/100 g-cassava meal, which was much lower than that in corn meal (7.90 g/100 g-corn meal). These data suggested that protein content, namely the nitrogen source content, is severely insufficient in cassava meal. Thus, it could be speculated that the lack of nitrogen source in cassava meal may cause the phase shift delay, and therefore leading to the fermentation performance deterioration. On the other hand, pH dropped down to and then stayed at a very low level (4.0–4.5) without rebound for a long time in the fermentations using cassava substrate. Therefore, it was considered that the consecutively low level of pH might be another reason accounting for the fermentation performance deterioration.
3.2. Accelerating phase shift and improving fermentation performance by addition of yeast extract From the above results, it is speculated that there are two possible reasons accounting for the delay of phase shift triggering when using cassava substrate. One is the ‘‘acid crash’’ effect due to the severe acetate and butyrate accumulation but without any re-assimilation. It was reported that when an ‘‘acid crash’’ happens, the environment that is essential for shifting from acidogenic phase to solventogenic phase is deteriorated, so that the excessively produced acids cannot be converted into solvents normally (Maddox et al., 2000). Another reason is the lower contents of nitrogen sources and other necessary elements in the medium as pointed out previously.
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F
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Fig. 1. Performance comparison of butanol fermentations using corn and cassava substrates. (A and B) pH and gas production. — / - - -: pH; ./5: gas production. (C and D) Butanol and residual starch concentrations. d/s: Butanol in aqueous phase; /e: butanol in extractant phase; N/4: residual starch. (E and F) Organic acids concentrations. j/ h: acetate; I/.: butyrate. (A and F) —: Cassava substrate; - - -: corn substrate. (A, C and E) Results of traditional butanol fermentation; (B, D and F) results of extractive butanol fermentation using oleyl alcohol as the extractant.
To deal with the potential ‘‘acid crash’’ problem, in ABE extractive fermentation using cassava substrate and with oleyl alcohol as the extractant, when pH continuously decreased without rebound and gas production tended to stop, ammonia water was added into broth to bring pH back to neutralized range (pH = 5.0–5.5) by force. However, after two attempts of adding ammonia water, as shown in Fig. 2A, pH continuously dropped down once again and gas production could not be recovered. As a result, it could be concluded that pH adjustment could not trigger the phase shift from acidogenic into solventogenic phase. Since regulating pH did not work, another rescue measure was taken by adding concentrated yeast extract solution (final concentration in medium was 2.5 g/L-broth) at 70 h of the fermentation. Yeast extract, enriched with nitrogen sources as proteins and amino acids, is one of the common used nitrogen sources for cell culture and fermentation processes. As shown in Fig. 2A, pH began to
rebound and gas production accelerated after addition of the concentrated yeast extract solution with only a 10 h response delay, and the phase shift occurred smoothly after that. As shown in Fig. 2B, final butanol concentrations in aqueous and extractant phases reached 8.04 and 27.34 g/L respectively, which were equivalent to those of using corn substrate. In addition, starch consumption was accelerated after adding the concentrated yeast extract solution, and starch was almost completely used out at the end of the fermentation. From the above results, it could be concluded that lower nitrogen contents in cassava substrate is the reason for the phase shift delay occurrence and the overall fermentation performance deterioration. To further verify the effectiveness of above mentioned manipulation and the relevant speculation, traditional and extractive fermentations (with oleyl alcohol as the extractant) using cassava substrate were conducted. Concentrated yeast extract solution
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X. Li et al. / Bioresource Technology 125 (2012) 43–51 Table 1 Amino acids concentrations in corn/cassava meal media and contents in corn/cassava meal after complete proteins hydrolysis. Medium
Concentrations of amino acids (g/L or g/100 g-meal) Total
His
Thr
Lys
Met
Tyr
Phe
Val
Leu
Corn
2.03E01a 7.90E+00b
4.27E03a 2.99E01b
1.94E05a 2.97E01b
2.77E03a 2.01E01b
5.67E02a 1.65E01b
5.57E03a 2.31E01b
5.51E03a 4.20E01b
4.40E04a 4.42E01b
3.30E03a 9.83E01b
Cassava
3.31E01a 1.81E+00b
6.24E03a 4.60E02 b
1.51E02a 6.84E02b
7.79E03a 6.38E02b
6.16E04a 4.95E02b
2.44E02a 4.53E02b
1.83E02a 7.59E02b
2.03E02a 1.16E01b
3.21E03a 1.23E01b
Cassava + yeast extractA
9.03E01a
1.67E02a
4.35E02a
4.65E02a
9.52E03a
6.60E02a
4.78E02a
7.11E02a
3.69E03a
His: histidine; Thr: threonine; Lys: lysine; Met: methionine; Tyr: tyrosine; Phe: phenylalanine; Val: valine; Leu: leucine. a Free amino acids concentrations in corn and cassava meal media after enzymatic pre-treatment (g/L). b Amino acids contents after complete proteins hydrolysis of corn or cassava meal (g/100 g-corn or cassava meal). A Concentration of yeast extract in the medium is 2.5 g/L-broth.
90
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0
0 60 80 100 120 140 160 180 Fermentation time (h)
Fig. 2. Fermentation course using cassava substrate subject to pH adjustment (at 34 and 56 h) and yeast extract addition (at 70 h). (A) pH and gas production. —: pH; .: gas production; B: butanol and residual starch concentrations. d: Butanol in aqueous phase; : butanol in extractant phase; N: residual starch.
(2.5 g/L-broth) was added during the phase shift delay period when gas production tended to cease. Fermentation performances were compared between the cases with and without yeast extract addition. As shown in Fig. 3A and B, after supplement of yeast extract for both traditional and extractive fermentations, the phase shift smoothly occurred about 10 h with pH rebounded whilst gas production recovered accordingly. Gas production, which is closely associated with the solvents production, increased significantly in fermentations with yeast extract addition as compared to those without. In clostridia metabolism, H2 and CO2 were the only two components in the evolved gas. H2 is generated in the electron transport
shuttle system by the catalysis of hydrogenase, and CO2 is generated in pyruvate ? acetyl-CoA and acetoacetyl-CoA ? acetone routes. When the acidogenic phase successfully shifts into the solventogenic phase, acetate and butyrate are re-assimilated and converted into acetyl-CoA and butyryl-CoA, respectively, accompanied with acetoacetyl-CoA converting into acetone. During this period, large amount of CO2 has to be released while H2 generation is decreased as butanol synthesis needs a large amount of NADH. Therefore, when the shift from acidogenic phase to solventogenic phase occurred, a reduced ratio of H2/CO2 could always be observed (Jones and Woods, 1986). As shown in Fig. 3A and B, when concentrated yeast extract solution was supplemented at 21 h during the traditional fermentation, H2/CO2 ratio was reduced rapidly from its top peak of 1.5 to a level lower than 0.25, and then lingered at low levels of 0.3–0.6 throughout the rest of fermentation period. For extractive fermentation case, H2/CO2 ratio also quickly decreased from 0.80 to lower levels of 0.25–0.55 after yeast extract addition at 39 h, and then stayed at these lower levels for about 25 h before going back to 0.80 again. In addition, the accumulated organic acids, acetate and butyrate, swiftly declined after yeast extract addition in both traditional and extractive fermentations, as shown in Fig. 3E and F. This suggested that acetate and butyrate are re-assimilated to form the precursors for butanol synthesis. Moreover, the two fermentation modes with the addition of concentrated yeast extract solution proceeded almost to completion whilst the concentrations of residual starch were reduced to lower level. As shown in Table 2 and Fig. 3C and D, When yeast extract was added in cassava meal medium during the delay period of phase shift in traditional fermentation, butanol productivity (0.27 g/L h1) was comparably higher than that of using corn substrate, and much higher (47–80%) than that of using cassava substrate but without yeast extract addition, as the phase shift delay was effectively shortened. At the same time, final butanol concentration (13.60 g/L) and butanol yield from starch (0.31 g/g-starch) were almost equivalent to those obtained with corn substrate. For the extractive fermentation with cassava substrate and yeast extract addition, total butanol concentration (34.37 g/L), butanol yield from starch (0.35 g/g-starch) and butanol productivity (0.34 g/L h1) were equivalent or even superior to those obtained with corn substrate. In particular, the selective butanol/acetone ratio, another very important performance index, strikingly reached 2.84, which is the highest among all fermentation operation modes. All these results pointed to a conclusion that timely and adequate addition of yeast extract could promote phase shift occurrence and improve fermentation performances comprehensively when using cassava substrate. The same operation procedure, timely and adequate addition of yeast extract, was also applied to the extractive fermentation with cassava substrate and using biodiesel as the extractant. In this case, butanol concentration in biodiesel, butanol yield from starch and
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Fig. 3. Performance comparison of butanol fermentation using cassava substrate with/without yeast extract addition during operation. (A and B) pH, H2/CO2 and gas production. — / - - -: pH; w: H2/CO2; ./5: gas production. (C and D) Butanol and residual starch concentrations. d/s: Butanol in aqueous phase; /e: butanol in extractant phase; N/4: residual starch. (E and F) Organic acids concentrations. j/h: Acetate; I/.: butyrate. (A–F): —: With yeast extract addition; - - -: without yeast extract addition. (A, C and E) Results of traditional butanol fermentation; (B, D and F) results of extractive butanol fermentation using oleyl alcohol as the extractant.
butanol productivity (10.56 g/L, 0.28 g/g-starch, 0.24 g/L h1) are all higher than those obtained with corn substrate (9.57 g/L, 0.25 g/g-starch, 0.20 g/L h1) and with cassava substrate but without yeast extract addition (8.16 g/L, 0.23 g/g-starch, 0.18 g/L h1). Butanol amount extracted in biodiesel, namely the butanol concentration in biodiesel, is a very important index reflecting the quality of the biodiesel. Higher butanol concentration in biodiesel directly leads to a higher biodiesel quality. In the case of using cassava substrate with yeast extract addition, butanol concentration in biodiesel reached a level of 10.56 g/L, which was 10% higher than that obtained with corn substrate, and this potentially contributed to the biodiesel quality improvement. The comparison results of fermentation performances under different conditions and operation modes were summarized in Table 2.
3.3. Effect of yeast extract supplement on genetic transcription level and accumulation of the amino acids stimulating butanol synthesis A typical feature of the clostridial solvent production is biphasic fermentation (Ezeji et al., 2007). The shift from acidogenic phase to solventogenic phase plays an important role during bio-butanol fermentation process, as the solvent products are formed smoothly only when the phase shift is triggered successfully. Currently, the factors triggering the metabolic shift are not clearly understood. Several investigations have reported that in batch cultures, the initiation of solvent production is associated with low extracellular and intracellular pH, as well as a high undissociated butyric acid concentration (Bahl et al., 1982; Monot et al., 1984). In this study, variations in genetic transcriptional levels of the key enzymes responsible for solvents production and major amino acids in broth
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X. Li et al. / Bioresource Technology 125 (2012) 43–51 Table 2 Fermentation performance under various operation modes with corn and cassava substrates. Batch #
Run time (h)
Final butanol in aqueous phase (g/L)
Total solvents (g/L) Butanol
Acetone
T1 T2 T3 T4 T5 T6 T7 E-OA1 E-OA2 E-OA3 E-OA4 E-OA5 E-OA6 E-BD1 E-BD2 E-BD3
65 68 80 51 60 38 22 102 100 92 162 101 99 90 83 78
11.63 13.74 11.80 13.60 12.95 4.41 1.27 8.19 6.21 3.54 8.04 7.07 6.60 8.80 6.57 7.81
11.63 13.74 11.80 13.61 12.95 4.41 1.27 35.87 27.15 19.04 35.38 34.37 29.81 18.37 (9.57a) 14.73 (8.16a) 18.37 (10.56a)
5.86 7.63 5.85 6.07 6.98 2.15 0.56 20.37 14.22 8.13 18.03 12.12 10.76 8.95 7.68 8.97
Butanol yield (g/gstarch)
Butanol productivity g/ (Lh)
Gas production amount (L/Lbroth)
0.25 0.27 0.27 0.31 0.28 0.10 0.08 0.23 0.26 0.26 0.22 0.35 0.29 0.25 0.23 0.28
0.18 0.20 0.15 0.27 0.22 0.12 0.06 0.35 0.27 0.19 0.22 0.34 0.30 0.20 0.18 0.24
23.26 37.36 17.72 39.71 29.85 11.62 7.67 86.13 78.96 34.35 81.56 77.74 60.09 32.87 26.36 42.66
T: Traditional fermentation runs; E-OA: Extractive fermentation runs with oleyl alcohol as the extractant; E-BD: Extractive fermentation runs with biodiesel as the extractant. T1-T2: corn substrate; T3: cassava substrate; T4–T5: cassava substrate and addition of yeast extract (2.5 g/L-broth) at 20 h and 16 h respectively; T6: corn substrate and addition of yeast extract (2.5 g/L-broth) at the beginning; T7: cassava substrate addition of yeast extract (2.5 g/L-broth) at the beginning. E-OA1–E-OA2: corn substrate; E-OA3–E-OA6: cassava substrate. E-OA3: normal run without yeast extract addition; E-OA4: adding ammonia water at 34 h and 55 h respectively and adding yeast extract at 71 h; E-OA5–E-OA6: adding yeast extract (2.5 g/L-broth) at 39 h. E-BD1: corn substrate; E-BD2: cassava substrate without yeast extract addition; E-BD3: cassava substrate and addition of yeast extract (2.5 g/L-broth) at 28 h. a Butanol concentration in bio-diesel.
during fermentations were analyzed or measured, attempting to explore the mechanism of how yeast extract addition promotes the phase shift and regulates butanol production. Nolling et al. (2001) proposed a metabolic map indicating the mechanism of butanol biosynthesis by C. acetobutylicum ATCC824. According to this map, the most important enzyme responsible for the phase shift from acidogenic to solventogenic phase is CoAtransferase which is encoded by ctfAB gene. CoA-transferase could re-assimilate the organic acids formed, and convert acetate and butyrate into acetyl-CoA and butyryl-CoA, the precursors of ethanol and butanol, respectively, whilst it could also convert acetoaceyl-CoA to acetoacetate, the precursor of acetone. On the other hand, butyraldehyde dehydrogenase (encoded by gene adhE) and butanol dehydrogenase (encoded by gene bdhB) are the two key enzymes directly associated with butanol biosynthesis. Here, the transcriptional levels of these genes during the extractive fermentations using oleyl alcohol as the extractant, with and without yeast extract supplement, were measured. 16S RNA in C. acetobutylicum ATCC 824 served as the internal reference. The gene transcriptional levels were quantified by PCR measurements. The normalized transcriptional levels (NTL) were then calculated based on Eq. (2) for the comparison and interpretation purposes, and the results were indicated in Fig. 4.
NTLjk ðiÞ ¼
TLjk ðiÞ Maxi;j fTLjk ðiÞg
i ¼ 1; N;
J ¼ 1; 2;
k ¼ 1; 3
ð2Þ
where TL is the measured (absolute) transcriptional levels, k represents the k-th gene (k = 1, 3; ctfAB, adhE or bdhB), j accounts for the comparison groups (j = 1, 2; with/without yeast extract addition), i refers to the ith measurement data (a total of N) within one comparison group and for one particular gene. As shown in Fig. 4A, the normalized transcriptional level of ctfAB, NTLctfAB, increased significantly and continuously from 0.063 to 1 with a maximal enhancement of 16-fold when yeast extract was supplemented at 39 h, while NTLctfAB without yeast extract addition remained at very low levels of 0.018–0.066 without any significant change. On the other hand, NTLadhE and
NTLbdhB, the normalized transcriptional levels of genes adhE and bdhB, varied irregularly for both cases (with and without yeast extract addition) during the fermentations, and no continuous and significant enhancements (>3-fold) were observed, as shown in Fig. 4B. These data indicated that the transcriptional level of ctfAB was considerably stimulated after yeast extract addition, and CoAtransferase was activated accordingly leading to smooth phase shift occurrence and acceleration of organic acids re-assimilation. CoA-transferase activation determined the butanol production enhancement after the yeast extract addition. Butyraldehyde dehydrogenase (encoded by adhE) and butanol dehydrogenase (encoded by bdhB) which are directly associated with butanol biosynthesis were not activated by yeast extract addition, thus they were not responsible for the butanol production enhancement. In cellular metabolism, amino acids are necessary for cell growth and also are important metabolic intermediates in ABE fermentation by C. acetobutylicum. It was reported that some amino acids, either self-generated or added, are favourable for butanol synthesis, but some others not. Masion et al. (1987) discovered that histidine has a beneficial effect on both solvents production and cell growth. Heluane et al. (2011) reported that lysine and methionine as butanol up-regulated products could enhance butanol production. Here in this study, we measured the major amino acids accumulated in cassava meal medium before and after yeast extract addition, to interpret the reason of butanol production enhancement after adding yeast extract from the other viewpoint. The results indicated that concentrations of histidine (histidine family), as well as threonine, lysine and methionine (aspartic acid family) in the broth increased remarkably during the period of 56– 66 h after yeast extract addition at 39 h, and then they decreased gradually (Fig. 5A). The maximal increments after yeast extract addition were 11-, 182-, 39- and 21-fold for histidine, threonine, lysine and methionine, respectively. Comparatively, when yeast extract was added into the fresh cassava meal medium, these amino acids only increased 3 (histidine), 3 (threonine), 6 (lysine) and 15-fold (methionine), as shown in Table 1. The huge increasing attitudes differences of these amino acids suggested that secre-
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X. Li et al. / Bioresource Technology 125 (2012) 43–51
A
A
0.10
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65
Fig. 4. Changes in transcriptional levels of ctfAB, adhE and bdhB with or without yeast extract addition (at 39 h) in extractive fermentations using cassava substrate. (A) Transcriptional level of ctfAB. White bars: without yeast extract addition; black bars: with yeast extract addition. (B) Transcriptional levels of adhE and bdhB. White bars: adhE, without yeast extract addition; black bars: adhE, with yeast extract addition; slashed shadow bars: bdhB, without yeast extract addition; parallel shadow bars: bdhB, with yeast extract addition.
tions of histidine, threonine, lysine and methionine were not brought by the contents of yeast extract itself, but by cellular production simulated by yeast extract addition. Then the over-produced histidine and aspartic acid families enhanced butanol production in turn. On the other hand, as shown in Fig. 5B, amino acids from aromatic family (including tyrosine and phenylalanine) and pyruvate family (valine and leucine), which were not reported to have positive effects on butanol synthesis, did not change significantly after yeast extract addition in cassava meal medium. From the above mentioned results, it could be concluded that addition of yeast extract during ABE fermentations on cassava substrate activated CoA-transferase and stimulated the secretions of histidine and aspartic acid families, thus, butanol productions were largely enhanced. Then, there is an argument whether addition of yeast extract into cassava or corn substrate at the beginning of the fermentations might also enhance ABE fermentation performance? To clarify the doubt, concentrated yeast extract solution (2.5 g/L-broth) was added at the beginning of traditional fermentations on both corn and cassava substrates. The results showed that fermentations ceased at about 30 h without phase shift occurring for both cases. Final butanol concentrations were very low (4.41 g/L for corn substrate case and 1.27 g/L for cassava substrate case, Table 2). In contrast, concentrations of acetate and butyrate were high without reduction until the end of fermentation (2.56
29 44 56 66 Fermentation time (h)
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Fig. 5. Changes of certain amino acids in cassava meal medium during extractive fermentation with yeast extract addition at 39 h. (A) The accumulations of histidine and aspartic acid families. Black bars: histidine (His); white bars: threonine (Thr); slashed shadow bars: lysine (Lys); parallel shadow bars: methionine (Met). (B) The accumulations of pyruvatic acid and aromatic acid families. Black bars: tyrosine (Tyr); white bars: phenylalanine (Phe); slashed shadow bars: valine (Val); parallel shadow bars: leucine (Leu).
and 1.34 g/L for corn substrate case, 2.34 and 0.62 g/L for cassava substrate case). With this operation mode, the normal metabolism on both corn and cassava substrates was destroyed and phase shift did not happen, suggesting that activation of CoA-transferase and over-secretions of amino acids would never occur in this case. These results demonstrated that yeast extract addition has the effects on promoting phase shift occurrence and enhancing fermentation performances using cassava substrate, when and only when it is added during the delay period. 4. Conclusions Cassava, a perspective substrate for bio-butanol production, was deficient in nitrogen source, leading to a severe phase shift delay and low butanol productivity. When yeast extract was added during the delay period of fermentation on cassava substrate, phase shift was triggered subsequently, and entire fermentation performances reached equivalent levels of those using corn substrate. Analysis of genetic transcriptional levels demonstrated that yeast extract could promote phase shift by largely increasing the transcription level of ctfAB to 16-fold. Moreover, yeast extract could stimulate secretions of amino acids from histidine and aspartic acid families, which could further enhance bio-butanol production.
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Acknowledgements The study was supported by National Natural Science Foundation Program (#20976072) and Major State Basic Research Development Program (#2007CB714303) of China. The authors also appreciated assistance from Mr. Jiang Xu and Mr. Martin D’Ambrosio in refining English of the manuscript. References Bahl, H., Andersch, W., Braun, K., Gottschalk, G., 1982. Effect of pH and butyrate concentration on the production of acetone and butanol by Clostridium acetobutylicum grown in continuous culture. Appl. Microbiol. Biotechnol. 14, 17–20. Bankar, S.B., Survase, S.A., Singhal, R.S., Granstrom, T., 2012. Continuous two stage acetone–butanol–ethanol fermentation with integrated solvent removal using Clostridium acetobutylicum B 5313. Bioresour. Technol. 106, 110–116. Cock, J.H., 1982. Cassava: a basic energy source in the tropics. Science 218, 755–762. Dhamole, P.B., Wang, Z., Liu, Y., Wang, B., Feng, H., 2012. Extractive fermentation with non-ionic surfactants to enhance butanol production. Biomass Bioenerg. 40, 112–119. Durre, P., 2007. Biobutanol: an attractive biofuel. J. Biotechnol. 2, 1525–1534. Evans, P.J., Wang, H.Y., 1988. Enhancement of butanol formation by Clostridium acetobutylicum in the presence of decanol-oleyl alcohol mixed extractants. Appl. Environ. Microb. 54, 1662–1667. Ezeji, T.C., Qureshi, N., Blaschek, H.P., 2007. Bioproduction of butanol from biomass: from genes to bioreactors. Curr. Opin. Biotech. 18, 220–227. Green, E.M., 2011. Fermentative production of butanol-the industrial perspective. Curr. Opin. Biotech. 22, 337–343. Gu, Y., Hu, S., Chen, J., Shao, L., He, H., Yang, Y., Yang, S., Jiang, W., 2009. Ammonium acetate enhances solvent production by Clostridium acetobutylicum EA 2018 using cassava as a fermentation medium. J. Ind. Microbiol. Biotechnol. 36, 1225–1232. Heluane, H., Evans, M.R., Dagher, S.F., Bruno-Barcena, J.M., 2011. Meta-analysis and functional validation of nutritional requirements of solventogenic clostridia growing under butanol stress conditions and coutilization of D-glucose and Dxylose. Appl. Environ. Microb. 77, 4473–4485. Jones, D.T., Woods, D.R., 1986. Acetone–butanol fermentation revisited. Microbiol. Res. 50, 484–524. Khan, S.A., Rashmi, Hussain, M.Z., Prasad, S., Banerjee, U.C., 2009. Prospects of biodiesel production from microalgae in India. Renew. Sust. Energ. Rev. 13, 2361–2372. Lee, S.Y., Park, J.H., Jang, S.H., Nielsen, L.K., Kim, J., Jung, K.S., 2008. Fermentative butanol production by clostridia. Biotechnol. Bioeng. 101, 209–228. Li, Z., Li, X., Zheng, J., Zhang, S., Shi, Z., 2011. Butanol extractive fermentation to simultaneously produce ‘‘properties improved’’ biodiesel and butanol in a water and energy-saving operation way. J. Biobased Mater. Bioenergy 5, 312–318. Lu, C., Zhao, J., Yang, S.-T., Wei, D., 2012. Fed-batch fermentation for n-butanol production from cassava bagasse hydrolysate in a fibrous bed bioreactor with continuous gas stripping. Bioresour. Technol. 104, 380–387.
51
Maddox, I.S., Steiner, E., Hirsch, S., Wessner, S., Gutierrez, N.A., Gapes, J.R., Schuster, K.C., 2000. The cause of ‘‘acid-crash’’ and ‘‘acidogenic fermentations’’ during the batch acetone–butanol–ethanol (ABE) fermentation process. J. Mol. Microb. Biotech. 2, 95–100. Masion, E., Amine, J., Marczak, R., 1987. Influence of amino acid supplements on the metabolism of Clostridium acetobutylicum. FEMS Microbiol. Lett. 43, 269–274. Monot, F., Engasser, J.M., Petitdemange, H., 1984. Influence of pH and undissociated butyric acid on the production of acetone and butanol in batch cultures of Clostridium acetobutylicum. Appli. Microbiol. Biotechnol. 19, 422–426. Nolling, J., Breton, G., Omelchenko, M.V., Makarova, K.S., Zeng, Q.D., Gibson, R., Lee, H.M., Dubois, J., Qiu, D.Y., Hitti, J., Wolf, Y.I., Tatusov, R.L., Sabathe, F., DoucetteStamm, L., Soucaille, P., Daly, M.J., Bennett, G.N., Koonin, E.V., Smith, D.R., Finishing, G.T.C.S.C.P., 2001. Genome sequence and comparative analysis of the solvent-producing bacterium Clostridium acetobutylicum. J. Bacteriol. 183, 4823–4838. Qureshi, N., Blaschek, H.P., 2001a. ABE production from corn: a recent economic evaluation. J. Ind. Microbiol. Biotechnol. 27, 292–297. Qureshi, N., Blaschek, H.P., 2001b. Evaluation of recent advances in butanol fermentation, upstream, and downstream processing. Bioproc. Biosyst. Eng. 24, 219–226. Qureshi, N., Ezeji, T.C., 2008. Butanol, ‘a superior biofuel’ production from agricultural residues (renewable biomass): recent progress in technology. Biofuel. Bioprod. Biorefin. 2, 319–330. Qureshi, N., Ezeji, T.C., Ebener, J., Dien, B.S., Cotta, M.A., Blaschek, H.P., 2008. Butanol production by Clostridium beijerinckii. Part I: use of acid and enzyme hydrolyzed corn fiber. Bioresour. Technol. 99, 5915–5922. Qureshi, N., Lolas, A., Biaschek, H.P., 2001. Soy molasses as fermentation substrate for production of butanol using Clostridium beijerinckii BA101. J. Ind. Microbiol. Biotechnol. 26, 290–295. Qureshi, N., Saha, B.C., Dien, B., Hector, R.E., Cotta, M.A., 2010a. Production of butanol (a biofuel) from agricultural residues. Part I – use of barley straw hydrolysate. Biomass Bioenerg. 34, 559–565. Qureshi, N., Saha, B.C., Hector, R.E., Dien, B., Hughes, S., Liu, S., Iten, L., Bowman, M.J., Sarath, G., Cotta, M.A., 2010b. Production of butanol (a biofuel) from agricultural residues: Part II – use of corn stover and switchgrass hydrolysates. Biomass Bioenerg. 34, 566–571. Roffler, S.R., Blanch, H.W., Wilke, C.R., 1987. In-situ recovery of butanol during fermentation. Bioproc. Biosyst. Eng. 2, 1–12. Taya, M., Ishii, S., Kobayashi, T., 1985. Monitoring and control for extractive fermentation of Clostridium acetobutylicum. J. Ferment. Technol. 63, 181–187. Thang, V.H., Kanda, K., Kobayashi, G., 2010. Production of acetone–butanol–ethanol (ABE) in direct fermentation of cassava by Clostridium saccharoperbutylacetonicum N1–4. Appl. Biochem. Biotech. 161, 157–170. Thirmal, C., Dahman, Y., 2012. Comparisons of existing pretreatment, saccharification, and fermentation processes for butanol production from agricultural residues. Can. J. Chem. Eng. 90, 745–761. Wang, Y., Blaschek, H.P., 2011. Optimization of butanol production from tropical maize stalk juice by fermentation with Clostridium beijerinckii NCIMB 8052. Bioresour. Technol. 102, 9985–9990. Zhang, L., Yang, Y., Shi, Z., 2008. Performance optimization of property-improved biodiesel manufacturing process coupled with butanol extractive fermentation. Chin. J. Biotechnol. 24, 1943–1948.