One-pot bioethanol production from cellulose by co-culture of Acremonium cellulolyticus and Saccharomyces cerevisiae
© Park et al.; licensee BioMed Central Ltd. 2012
Received: 4 June 2012
Accepted: 30 August 2012
Published: 31 August 2012
While the ethanol production from biomass by consolidated bioprocess (CBP) is considered to be the most ideal process, simultaneous saccharification and fermentation (SSF) is the most appropriate strategy in practice. In this study, one-pot bioethanol production, including cellulase production, saccharification of cellulose, and ethanol production, was investigated for the conversion of biomass to biofuel by co-culture of two different microorganisms such as a hyper cellulase producer, Acremonium cellulolyticus C-1 and an ethanol producer Saccharomyces cerevisiae. Furthermore, the operational conditions of the one-pot process were evaluated for maximizing ethanol concentration from cellulose in a single reactor.
Ethanol production from cellulose was carried out in one-pot bioethanol production process. A. cellulolyticus C-1 and S. cerevisiae were co-cultured in a single reactor. Cellulase producing-medium supplemented with 2.5 g/l of yeast extract was used for productions of both cellulase and ethanol. Cellulase production was achieved by A. cellulolyticus C-1 using Solka-Floc (SF) as a cellulase-inducing substrate. Subsequently, ethanol was produced with addition of both 10%(v/v) of S. cerevisiae inoculum and SF at the culture time of 60 h. Dissolved oxygen levels were adjusted at higher than 20% during cellulase producing phase and at lower than 10% during ethanol producing phase. Cellulase activity remained 8–12 FPU/ml throughout the one-pot process. When 50–300 g SF/l was used in 500 ml Erlenmeyer flask scale, the ethanol concentration and yield based on initial SF were as 8.7–46.3 g/l and 0.15–0.18 (g ethanol/g SF), respectively. In 3-l fermentor with 50–300 g SF/l, the ethanol concentration and yield were 9.5–35.1 g/l with their yields of 0.12–0.19 (g/g) respectively, demonstrating that the one-pot bioethanol production is a reproducible process in a scale-up bioconversion of cellulose to ethanol.
A. cellulolyticus cells produce cellulase using SF. Subsequently, the produced cellulase saccharifies the SF, and then liberated reducing sugars are converted to ethanol by S. cerevisiae. These reactions were carried out in the one-pot process with two different microorganisms in a single reactor, which does require neither an addition of extraneous cellulase nor any pretreatment of cellulose. Collectively, the one-pot bioethanol production process with two different microorganisms could be an alternative strategy for a practical bioethanol production using biomass.
KeywordsBioethanol Cellulase Biomass Acremonium cellulolyticus C-1 Saccharomyces cerevisiae Biorefinery
A co-culture as a mimic of natural environment has been used for biodegradation of aromatic compounds[1, 2] or for biological reduction of sulfate. In addition, ethanol production from a mixture of glucose and xylose was applied using co-cultures of Pichia stipites with Zymomonas mobilis or Saccharomyces cerevisiae. Thus the co-culture is a potential bioprocess if there are no cross-interactions among microorganisms, and each microorganism metabolizing its substrate is unaffected by the presence of other microorganism.
To prepare lignocellulose for ethanol production, the substrate is normally either hydrolysed completely to the reducing sugars using mineral acids or solubilized with a milder pretreatment, leaving the residual cellulose to be saccharified enzymatically. For a conversion of this cellulose to ethanol, either diluted-acid hydrolysate or cellulase-saccharified hydrolysate was used for the co-culture using yeast with Escherichia coli or Z. mobilis, respectively. When acid-hydrolysate is used, detoxification of the inhibitory components is required to increase the yield. In addition, when glucose and xylose are converted by co-culture using different microorganisms to ethanol production, the saccharification process is still essentially required. Alternatively a one-step process, a combination of cellulase production, cellulose hydrolysis, and fermentation, was applied in the co-culture of Clostridium thermocellum and Z. mobilis in 10 ml scale. From this co-culture, 2.7 mg/ml of ethanol was produced from 10 mg/ml of cellulose. However, C. thermocellum was significantly inhibited at the low levels of ethanol, and leaving the undegraded cellulobiose in the co-culture inhibited the cell-associated cellobiase, which prevented the efficient conversion of cellulose to ethanol.
Recently, many researches have focused on the consolidated bioprocessing (CBP) for the simplification of the conversion process of cellulose to bioethanol[9, 10]. The CBP was categorized into CBPs I and II. Category I CBP is an engineering method of a cellulase producer to be ethanologenic, while category II CBP of an ethanologen to be cellulolytic. A prototype model of CBP I is Trichoderma reesei or C. thermocellum, which is one of the widely studied microorganisms because of producing several kinds of cellulases and β-glucosidases. These microorganisms can produce ethanol from cellulose, followed by the fermentation of the resulting sugars to ethanol in anaerobic growth conditions. However, its ethanol yield, productivity, and ethanol tolerance are low due to the low expression of the relevant genes involved in ethanol fermentation or to the low activity of the enzymes encoded by these genes. These bottlenecks have to be solved to improve the feasibility of the CBP I microorganism.
The CBP II strain requires functional production and secretion of the variety of exoglucanases and endoglucanases, assimilation and fermentation on lignocellulose as a sole carbon source. Target microorganisms are engineered E. coli or Z. mobilis in bacteria, and S. cerevisiae in yeasts. Cellulolytic enzymes have been functionally expressed in some of yeasts, but the yeast has not been satisfied in anaerobic growth on cellulose. Recently, cellulases, xylanases, and amylases were expressed on the cell surface of S. cerevisiae[14–16] and 2.1 g/l of ethanol was produced from 10 g/l of phosphate-swollen amorphous cellulose using endoglucanase, cellobiohydrolase, and β-glucosidase displaying yeast. Unfortunately, the expression of T. reesei cellobiohydrolases or exoglucanase, which play the critical role in cellulose degradation, is still poor in S. cerevisiae, and still remains lots of problems to be solved before a practical contribution to a worldwide energy supply.
In this study, one-pot bioethanol production system consisting of cellulase production from cellulose, saccharification of cellulose using cellulase in situ, and ethanol production was investigated. For one-pot ethanol production, understanding the concept of potential microorganisms to produce cellulase from cellulose and ethanol from hydrolysate is indispensable. Filamentous fungus Acremonium cellulolyticus, isolated in 1987, was used in this study. The A. cellulolyticus produced cellulase from crystal cellulose, pretreated waste milk pack, and untreated waste paper sludge (PS). The cellulase activities of A. cellulolyticus were comparable to those of T. reesei origins, which was enough to proceed the bioconversion of waste office paper to L(+)-lactate and gluconic acid. For example, 160 g/l of cellulose contained in the waste PS was successfully converted to 40 g/l of ethanol in SSF using this cellulase and thermotolerant S. cerevisiae. However, this kind of process is composed of two different and separate processes, such as cellulase and ethanol productions. A new emerging challenge of the one-pot ethanol process is to produce ethanol from cellulose in a single reactor using the co-culture of A. cellulolyticus and S. cerevisiae. A. cellulolyticus cells produce cellulase from cellulose, and the produced cellulase in situ saccharifies cellulose. S. cerevisiae cells consume the liberated reducing sugars and produce ethanol. Therefore, the combination of these different microorganisms has potentials for cellulase and ethanol productions.
This study is the first challenge for a practical application of ethanol production from cellulose in a single bioreactor using A. cellulolyticus and S. cerevisiae cells. A. cellulolyticus and S. cerevisiae cells grow in different media and different oxygen consumption for each other. For successful one-pot process for ethanol production, medium composition, times for inoculum of each microorganism and substrate addition should be carefully considered. This study demonstrated the high yield of ethanol production from biomass by optimizing these critical variables in one-pot bioethanol production.
S. cerevisiae inoculum time on co-culture of A. cellulolyticus and S. cerevisiae in a shake flask.
Medium preparation for one-pot bioethanol production in a shake flask
When both A. cellulolyticus and S. cerevisiae cells were co-cultured in a single reactor, DCWS was at low levels (Additional file1: Figure S1). Although cellulase production was followed by S. cerevisiae inoculum, DCWS was still at low levels (Figure2B). It means that it is difficult to get efficient production of ethanol in one-pot because of slow growth of S. cerevisiae due to depletion of nutrients. To promote the growth of S. cerevisiae, 0–5 g/l of yeast extract, 0–10 g/l of polypeptone, and their combined nutrients were added to cellulase-producing medium, respectively (Additional file2: Figure S2). A. cellulolyticus cell growth was similar (Additional file2: Figure S2 A) but cellulase activity was maintained higher than that without its addition (Additional file2: Figure S2 B), suggesting that yeast extract and polypeptone were not inhibitory to the cellulase production in this co-culture. Ethanol production was increased to 40% by addition of 5 g/l of yeast extract compared to that without its addition (Additional file2: Figure S2 C). Minimum requirement of yeast extract concentration for bioethanol production in S. cerevisiae was tested in the range of 0–7.5 g/l addition (Additional file3: Figure S3). A. cellulolyticus cell growth and cellulase production were not affected by yeast extract addition (Additional file3: Figure S3 A and B). The growth of S. cerevisiae was improved by 2–3 fold compared to that without its addition (Additional file3: Figure S3 C). Since significant effect on ethanol production was not observed (Additional file3: Figure S3 D), 2.5 g/l of yeast extract was added to cellulase-producing medium for stable growth of S. cerevisiae in the co-culture of two microorganisms. Thus, the minimal supplement of yeast extract significantly promoted the ethanol production as well as the growth of S. cerevisiae.
Effect of temperature on cellulase production
Effect of agitation rate on the productions of cellulase and ethanol in co-culture
One-pot bioethanol production from SF in a shake flask
To improve the ethanol concentration, initial SF was 150 g/l at 60 h and another 150 g SF/l was added during ethanol fermentation at 72, 78, and 84 h (Figure5C and D). The cellulase activity was 9–11.5 FPU/ml (Figure5C), which was not affected by addition of SF during the ethanol production. Ethanol concentration was 26.7 g/l from 150 g SF/l, but it increased to 45–46.3 g/l by addition of 150 g/l SF (Figure5D). Y e/SF s for addition 150 g SF/l at 72, 78, and 84 h were the same values of 0.15 g/g, respectively; overall ethanol production rate (Ve), 0.45–0.48 g/l/h. This result indicates that cellulase activity remained enough to saccharify SF in the ethanol production phase and simultaneously S. cerevisiae cells were active in the ethanol fermentation phase.
Improved one-pot bioethanol production from SF in fermentor
SF concentration was added another 150 g/l at the culture time of 66 h as the same cultural condition as Figure6A and B. When the agitation rate was decreased to 200 rpm at the culture time of 60 h, the dissolved oxygen level was dropped to 0%, but increased gradually as high as 20% at the culture time of 66 h (Figure6C). Cellulase activity was 12 FPU/ml at maximum and remained 10 FPU/ml during the ethanol production phase (Figure6D). The ethanol concentration was increased sharply reaching 35.1 g/l (Figure6D) at the culture time of 72 h, and the Y e/SF and Ve values were 0.12 g/g and 0.49 g/l/h, respectively. Thus, one-pot bioethanol production from cellulose by two microorganisms is applicable to a jar fermentor scale production platform.
While the CBP is considered as the most ideal process, CBP is not useful in practice due to low ethanol concentration and its low yield. Alternatively, a simultaneous saccharification and fermentation (SSF) is an appropriate method for bioethanol production, but, in SSF, cellulase production process is excluded. Previous study reported that the ethanol concentration and yield based on initial cellulose were 36.5 g/l and 0.25 (g ethanol/g PS cellulose) from 150 g/l of cellulose contained in PS, respectively with cellulase loading of 15 FPU/g PS cellulose. In the SSF, the total reaction time was 80 h, but when cellulase production process is included, it was at least 140 h, meaning that the Ve was 0.26 g/l/h.
In this one-pot bioethanol production process the productions of cellulase and ethanol were carried out by A. cellulolyticus and S. cerevisiae, respectively, in a single reactor. The total reaction time was dramatically reduced to 72 h including cellulase production, saccharification, and ethanol fermentation. During this process, the cellulase activity was not deactivated and remained constant (Figures5B,6B and D), enough to saccharify SF. The ethanol concentration was 25.6 g/l from 150 g SF/l with the Y e/SF of 0.17 g/g, which was 70% comparing to that of the SSF. However, the Ve was 0.36 g/l/h, which is 1.4 fold to that of SFF. When 300 g/l SF was used in flask with cellulase loading of 25 FPU/g SF to increase ethanol concentration, the produced ethanol concentration, Y e/SF , and Ve were 46.3 g/l, 0.15 g/g, and 0.48 g/l/h, respectively (Figure5D). However, in jar-fermentor, ethanol concentration, Y e/SF , and Ve were 35.1 g/l, 0.12 g/g, and 0.49 g/l/h, respectively (Figure6D).
The Ve was improved 1.4 – 1.9-folds to that of SSF, indicating that the cellulase produced from A. cellulolyticus was highly stable and remained high activity without deactivation. The cellulase activity of firstly isolated A. cellulolyticus was only 5.0 FPU/ml using cellulose powder. However, this activity was still insufficient for the saccharification of cellulolytic biomass industrially. We improved the cell line and optimized its medium for the practical production of cellulase and obtained 15.5 FPU/ml in the flask culture, 17.42 FPU/ml in the 7-L bioreactor, and 13.08 FPU/ml in the 50-L bioreactor. A. cellulolyticus produces a complex mixture of cellulases, mainly comprised of four β-glucosidases (EC 126.96.36.199) and twelve distinct endo-cellulases/carboxymethyl cellulases (CMCases, EC 188.8.131.52)[18, 25]. Other polysaccharide hydrolyzing enzymes, such as xylanases, amylases and β-1,3-glucanases are also present. The most important enzyme in this mixture for the current process is an endo-cellulose type III-A that can produce glucose from cellulose without any participation of β-glucosidase. In contrast, it is well known that the Trichoderma enzymes do not effectively saccharify cellulose alone because of their low β-glucosidase activity. When A. cellulolyticus cellulase and Trichoderma enzymes, GC220 were compared for saccharification of waste paper, their glucose contents among the hydrolysates were 83 and 72%, respectively. Because the genus of Trichoderma generally produces relatively low β-glucosidase in its cellulolytic enzymes, those enzymes cannot convert cellulolytic biomass to glucose efficiently without addition of extraneous β-glucosidase. It is very important to have higher glucose contents in the hydrolysate since it is only glucose that S. cerevisiae can easily uptake.
Although this one-pot bioethanol production process significantly improve overall ethanol production rate, low Y e/SF is still remained as an issue to be resolved. In the ethanol production phase A. cellulolyticus and S. cerevisiae cells consume glucose both for productions of cellulase and ethanol, respectively, and for their cellular maintenances, which cause the Y e/SF decreased. It is better to keep in anaerobic condition in the ethanol production phase, but it was necessary to some extent agitation rate to avoid a precipitation of SF inside the reactor. To keep anaerobic condition, it may be effective to purge a nitrogen gas, but A. cellulolyticus cells cannot be alive. In this experiment, the dissolved oxygen level in the ethanol production phase increased to 20%, which might decrease the carbon flux from glucose to ethanol. It is necessary to optimize the dissolved oxygen both for maximizing ethanol production and for maintaining A. cellulolyticus cells actively. So far, this one-pot bioethanol production is an alternative strategy as a mimic of CBP, because cellulase production, saccharification of carbohydrate, and ethanol fermentation occur in a single reactor.
This study establishes a method for practical one-pot bioethanol production from SF neither addition of extraneous cellulase nor pretreatment of cellulose. This one-pot bioethanol production includes cellulase production by A. cellulolyticus, saccharification of cellulose by cellulase in situ, and then ethanol production by S. cerevisiae from liberated reducing sugars in a single reactor. The potential of this process was also demonstrated in a stable and practical biorefinery using cellulosic biomass. Control of operational parameters, dissolved oxygen level, cellulose addition time, and S. cerevisiae inoculum time are still important for improving ethanol production in the co-culture with two different microorganisms. In particular, maintaining as higher than 20% and lower than 10% dissolved oxygen levels at cellulase and ethanol production phases, respectively, could be crucial for maximizing the one-pot bioethanol production. Further studies are planned to allow the improved ethanol yield from different cellulosic biomass and to be scaled up.
Solka-Floc (SF; CAS #9004-34-6; International Fiber Co., New York, USA) was used for cellulase and ethanol production. SF is a fine white powder comprised of approximately 70-80% crystalline cellulose and 20-30% amorphous cellulose. Medium components and other chemicals were purchased from Wako Pure Chem. Co. Ltd. (Tokyo, Japan) and stored at a room temperature.
A. cellulolyticus C-1 (Ferm P-18508), which is a hyper cellulase producer, and a mutant of wild type A. cellulolyticus Y-94, was provided by Tsukishima Kikai Co. Ltd. (Tokyo). S. cerevisiae ATCC 4126 (American Type Culture Collection, University Boulevard Manassas, VA, USA) was used for ethanol production in co-culture with A. cellulolyticus C-1.
The preculture medium for A. cellulolyticus is consisted (per liter) of 40 g SF, 24 g of KH2PO4, 1 ml of Tween 80 (MP Biomed. Co. Ltd., OH, USA), 5 g of (NH4)2SO4, 4.7 g of K2C4H4O6·4H2O, 1.2 g of MgSO4·7H2O, 10 mg of ZnSO4·7H2O, 9.28 mg of MnSO4·7H2O, 8.74 mg of CuSO4·7H2O and 2 g of urea (pH 4.0). The medium was sterilized at 121°C for 20 min, with separately sterilized ZnSO4·7H2O, MnSO4·7H2O and CuSO4·7H2O. Urea was sterilized by filtering through a 0.45 μm filter membrane (Toyo Roshi Kaisha Co. Ltd., Tokyo, Japan).
The preculture medium for S. cerevisiae is consisted (per liter) of 50 g glucose, 50 g/l YPD medium containing less than 0.04% of adenine (Sigma-Aldrich Co. Ltd., St. Louis, MO, USA). The YPD medium was composed of 20 g/l of bacteriological peptone, 10 g/l of yeast extract and 20 g/l of glucose.
Co-culture of A. cellulolyticus and S. cerevisiae
Seed cultivation of A. cellulolyticus was carried out using 5 ml medium in a test tube at 28°C and 220 revolution per min (rpm) for 65 h. Cellulase production was carried out in 500 ml Erlenmeyer flask with 50 ml medium at 28°C and 220 rpm after addition of A. cellulolyticus inoculum with its volume fraction of 5%(v/v). The seed culture of S. cerevisiae was incubated for 24–30 h and by the time the cell density was about 2.2–2.8 g dry cell weight (DCW)/l. Co-culture was begun by addition of the desired inoculum sizes of S. cerevisiae and desired amount of SF into the culture of A. cellulolyticus at different addition times. The SF as a powder was used for a cellulase-inducing substrate and was steam-sterilized before use.
Culture conditions of one-pot bioethanol production in a shake flask
To investigate medium components, culture temperature, agitation rate, and addition of S. cerevisiae inoculum for ethanol production in one-pot, co-culture was carried out in 500 ml Erlenmeyer flask with 50 ml medium. Medium is an important factor for producing both ethanol and cellulase with two different microorganisms. Preliminary test revealed yeast extract and polypeptone were the most effective nutrient at pH 4.5 and 28°C. Medium for A. cellulolyticus cultivation was added yeast extract, 0–7.5 g/l and used for productions of cellulase and ethanol. The effect of the culture temperature on cellulase production by A. cellulolyticus was tested in the temperature range of 24–44°C. To investigate the addition time of S. cerevisiae inoculum and SF for ethanol production, S. cerevisiae inoculum and SF were added at four different cellulase production phases, which was 48, 60, 72, and 96 h. A. cellulolyticus and S. cerevisiae requires different oxygen level, in aerobic and anaerobic growth pattern, respectively. Cellulase production in the culture of A. cellulolyticus was carried out in 500-ml Erlenmeyer flasks in a rotary shaker at an agitation rate of 220 rpm, but ethanol production in co-culture with S. cerevisiae with SF was carried out at different agitation rates of 80–220 rpm. After the addition of S. cerevisiae inoculum and SF, ethanol concentration and cellulase activity were measured.
One-pot bioethanol production in a jar fermentor
Three liters jar fermentor (MDL-80, Marubishi, Tokyo Japan) with a 1.67 l working volume was used. One hundred and fifty millilitres of seed culture of A. cellulolyticus were added to 1350 ml of cellulase production medium supplemented with nutrients, and then cultured at agitation rate of 500 rpm and aeration rate of 1.5 volume per volume per min (vvm). Co-culture was begun by adding a 10%(v/v) inoculum of S. cerevisiae and desired amount of SF as a powder into the culture of A. cellulolyticus at the desired culture time. After the addition of S. cerevisiae inoculum and SF, agitation rates decreased to 200 revolution per min (rpm). After culture, DCW of A. cellulolyticus (DCWA) and S. cerevisiae (DCWS) was measured. The culture broth was centrifuged at 9447 g, and the supernatant was stored in a 4°C refrigerator before the measurement of a cellulase activity and ethanol and protein concentrations.
where, ΔC e and C SF denote concentrations of produced ethanol and initial SF, respectively, and tone-pot indicates culture time including cellulase production, saccharification, and fermentation.
where, DCWS denotes DCW of S. cerevisiae.
Cellulase activity was measured using the standard IUPAC procedure with Whatman No. 1 filter paper, and the activity was expressed in filter paper unit (FPU). The FPU unit is based on the International Unit (IU) in which the absolute amount of glucose at a critical dilution is 2 mg for 0.5 ml critical enzyme concentration for 60 min.
Reducing sugar was measured by dinitrosalicylic acid (DNS). Residual glucose concentration was measured by a biochemistry analyzer (2700 SELECT, YSI Life Sci., Yellow Springs, OH, USA).
Ethanol concentration was measured using a Gas Chromatography (Shimadzu-2014, Shimadzu Co. Ltd., Tokyo, Japan) using a packed column (Gaskuropack5460/80, GC−2014Glass ID.3.2ϕ × 2.1 m, GL Science Co. Ltd., Tokyo, Japan), with the following operational conditions: temperature of column and detector were 110 and 250°C, respectively, nitrogen gas flow rate 60 ml/min and the injected sample volume 2 μl.
The total soluble protein concentration in crude and enzyme solutions was measured by the Lowry method. Protein standard was prepared using bovine serum albumin.
Statistical analysis of experimental data was performed by a factorial ANOVA, using the least significant difference by the STATISTICA software package (Ver 5.5, Tulsa, OK, USA).
analysis of variance
dry cell weight
dry cell weight of A. cellulolyticus
dry cell weight of S. cerevisiae
dry cell weight of A. cellulolyticus and S. cerevisiae
simultaneous saccharification and fermentation: Ve, overall ethanol production rate in one-pot bioethanol production process (g/l/h): Y e/SF , the ethanol yield based on the initial SF (g ethanol/g SF).
We thank to Dr. Naoyuki Okuda of Tsukishima Kikai Co. Ltd. (Tokyo, Japan) for kindly providing A. cellulolyticus C-1 strain for this study.
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