Cellulosic Ethanol Production by Engineered Consortia of Scheffersomyces Stipitis and Zymomonas Mobilis


 Background: As one of the clean and sustainable energies, lignocellulosic ethanol has achieved much attention around the world. The production of lignocellulosic ethanol does not compete with people for food, while the consumption of ethanol could contribute to the carbon dioxide emission reduction. Two of the conditions that are needed to attain cost-efficient lignocellulosic ethanol production at an industrial scale are the simultaneous transformation of glucose and xylose to ethanol and a highly efficient ethanol fermentation process. Results: In this study, the consortia consisting of suspended Scheffersomyces stipitis CICC1960 and Zymomonas mobilis 8b were cultivated to successfully depress carbon catabolite repression (CCR) in 80G40XRM. With this strategy, a 5.52% more xylose consumption and a 6.52% higher ethanol titer were achieved by the consortium, in which the inoculation ratio between S. stipitis and Z. mobilis was 1:3, at the end of fermentation compared with the Z. mobilis 8b mono-fermentation. Subsequently, one copy of the xylose metabolic genes was inserted into the Z. mobilis 8b genome to construct Z. mobilis FR2, leading to the xylose final-consumption amount and ethanol titer improvement by 15.36% and 6.81%, respectively. Finally, various concentrations of corn stover hydrolysates, in which the sum of glucose and xylose concentrations in the hydrolysates were 60, 90, and 120 g/L respectively, were used to evaluate the fermentation performance of the consortium consisting of S. stipitis CICC1960 and Z. mobilis FR2. Fermentation results showed that a 1.56% - 4.59% higher ethanol titer was achieved by the consortium compared with the Z. mobilis FR2 mono-fermentation, and a 46.12% - 102.14% higher ethanol titer was observed in the consortium fermentation when compared with the S. stipitis CICC1960 mono-fermentation. Conclusions: The fermentation strategy used in this study, i.e., using a genetically modified consortium, had a superior performance in ethanol production, as compared with the S. stipitis CICC1960 mono-fermentation and the Z. mobilis FR2 mono-fermentation alone. Thus, this strategy has potential for future lignocellulosic ethanol production.


Background
Lignocellulosic biomass, like hardwood, softwood, and grasses, is produced by plant photosynthesis from solar energy and is the most abundant renewable feedstock in the world. The bio-conversion of lignocellulosic biomass into ethanol by microbial fermentation is viewed as one of the most promising ways to partially replace traditional fossil fuels, since the combustion of ethanol produces less particulate matter, carbon monoxide, and hydrocarbons than fossil fuels (1). Additionally, as bioethanol has a high octane number, mixing bioethanol with gasoline could improve the anti-detonating quality of transportation fuel.
Lignocellulosic biomass is primarily degraded into glucose and xylose after pretreatment and enzymatic hydrolysis. The simultaneous and e cient bioconversion of the two sugars into ethanol are two of the prerequisites for large-scale production of cellulosic ethanol. However, due to carbon catabolite repression (CCR), a considerable amount of wild microbes and engineered microbes having exogenous xylose-metabolic pathways, like Zymomonas mobilis, Escherichia coli, Saccharomyces cerevisiae and Bacillus amyloliquefaciens, prefer to use glucose, and therefore their xylose utilization generally lags behind glucose utilization (2)(3)(4)(5). This greatly hampers the large-scale application of cellulosic ethanol in industry.
Extensive studies have been attempted to relax CCR. For example, as all known xylose transporters are suppressed by glucose, many researchers have tried to engineer glucose-insensitive xylose transporters by evolutionary engineering, error-prone PCR, and site-directed mutagenesis. By this way, researchers have successfully built Gal2-N376F, CiGXS1 FIVFH497* and AN25-R4.18 (6-8). In addition, adaptive evolution, computation simulation, and rational design have been used to nd appropriate intracellular targets to alleviate CCR, such as the phosphoenolpyruvate transferase system (PTS), the cyclic AMP receptor protein (CRP), and the xylose operon regulatory protein (9,10). However, the complex nature of CCR makes it di cult for us to entirely unveil how CCR functions. Moreover, those engineered strains developed by the abovementioned methods typically cannot co-utilize glucose and xylose with high e ciency (9).
An alternative method is to build arti cial consortia to co-ferment glucose and xylose. A common practice is to build a consortium consisting of a xylose-speci c strain due to the de ciency in the PTS system and a wild strain that utilizes glucose because of CCR (9). In this way, for instance, 20.82 g/L butanol with a yield of 0.35 g/g was produced from glucose and xylose using two E. coli strains. These results were comparable with butanol titers and yields produced in previous studies from glucose alone (11). Another strategy is to use two wild species to ferment the glucose and xylose mixture. For example, when Scheffersomyces stipitis and S. cerevisiae were co-fermented together, the xylose removal e ciency and ethanol production showed remarkable improvement than in their mono-fermentation alone (12). In comparison with two-stage fermentation (glucose and xylose are consumed in a separate fashion), the consortium fermentation was advantageous in terms of assimilating glucose and xylose concomitantly and shortening fermentation time.
Z. mobilis is an excellent ethanol-producing species whose ethanol production e ciency can reach as high as 98%, which is higher than S. cerevisiae (13). However, the wild Z. mobilis cannot utilize xylose unless it has been transformed by the exogenous xylose metabolic pathway, like Z. mobilis 8b (14). Additionally, S. stipitis is recognized as one of the best microbes in nature in terms of its xylose assimilation ability, but it has a severe CCR phenomenon. In recent studies, the consortium of S. stipitis and Z. mobilis has been studied for the co-fermentation of glucose and xylose. However, these studies generally involved two-stage fermentation, or the total sugar concentrations in lignocellulosic hydrolysate medium were low, which is unrealistic in large-scale fermentation (15)(16)(17)(18). Even for synthetic medium containing pure sugars, the xylose removal e ciency still requires improvement (19).
The present work focuses on investigating the potential of consortium fermentation consisting of S. stipitis and Z. mobilis in a synthetic medium and corn stover hydrolysate, with a great emphasis on alleviating CCR, increasing the sugar removal e ciency, and increasing the bioethanol production. The corn stover was chosen since it is one of the most common annual agricultural wastes produced in China (20). Instead of wasting the natural resource by burning it, it is better to transform it into useful products, like ethanol, and contribute to global warming mitigation.

Results And Discussion
2.1 Co-culture of S. stipitis CICC1960 and Z. mobilis 8b in 80G40XRM Arti cially synthesized 80G40XRM (80 g/L glucose + 40 g/L xylose) was rst used to explore the appropriate mode of this consortium fermentation with the aim of improving the glucose-and-xylose coutilization e ciency. The ratio between glucose and xylose in this medium was 2:1, aiming to simulate the real ratio in lignocellulosic hydrolysates.
In a previous study (21), when S. stiptis was pre-cultured in the medium with glucose as the sole carbon source (glucose medium), its xylose-metabolic gene expression, such as D-xylose reductase and xylitol dehydrogenase expression, was inhibited. Hence, when S. stiptis was inoculated in the xylose medium (xylose was the sole carbon source in the fermentation medium) later, its xylose-metabolic genes needed to be synthesized from scratch. Therefore, S. stiptis exhibited an apparent lag in fermentation in the xylose medium. In contrast, when S. stiptis was precultured in the xylose medium, the two xylosemetabolic genes were fully expressed and the aforementioned problem of lag in the xylose fermentation was alleviated, especially if S. stiptis was inoculated with high initial density, such as A 620 = 40. To this end, we decided to preculture S. stiptis CICC1960 in YP120X (120 g/L xylose) and then inoculate it to the 80G40XRM (fermentation medium) with a 100% (v/v) inoculum size, hoping to alleviate the CCR phenomenon in S. stiptis. However, as shown in Fig. 1a and 1b, S. stipitis CICC1960 still could not assimilate glucose and xylose at the same time. This might have been a result of the glucose repression on xylose transportation into the cells (6), or the inoculum size used in this study (the initial OD 600 in fermentation was about 1.8) was not high enough. For economic reasons, the inoculum size was not further increased in this study.
For Z. mobilis 8b, 80G40XRM was used as its seed culture medium, since Z. mobilis 8b cannot grow well in xylose medium (data not shown). The results of the Z. mobilis 8b mono-fermentation are shown in Fig. 1 and Additional le 1: Table S1. During fermentation, Z. mobilis 8b showed a strong ability to utilize glucose and xylose simultaneously with ethanol productivity reaching 5.10 g/L/h. More specially, the CCR in Z. mobilis 8b was largely alleviated, which may be attributed to the high inoculum size used in this study (the initial OD 600 was approximately 1.8). In 2004, Ali Mohagheghi et al. found that Z. mobilis 8b (the initial inoculum density was 0.2 at 600 nm) exhibited an apparent lag of xylose utilization, while glucose could be assimilated immediately (22). Since the xylose-metabolic genes were inserted to the genome of Z. mobilis 8b, their expression was generally low in individual cells. When Z. mobilis 8b were inoculated with high density, activities of those enzymes encoding xylose-metabolic genes would be higher than the ones under a low initial density inoculation, and this may contribute to the alleviation of CCR in this study (14). Glucose was completely removed within six hours by Z. mobilis 8b, while xylose still remained at 6.16 g/L at 21 h. The ethanol yield of the Z. mobilis 8b mono-fermentation was 0.48 g/g (the theoretical ethanol yield is 0.51 g/g sugars).
When S. stipitis CICC1960 and Z. mobilis 8b were co-cultured together with different inoculation ratios (3:1-1:3), the glucose consumption pro les of the consortia did not differ considerably from the Z. mobilis 8b mono-fermentation (Fig. 1a). Glucose was completely removed within nine hours by the consortia. On top of this, no apparent CCR was shown since xylose was rapidly assimilated at the very early stage (Fig. 1b). Although xylose consumption rates by the consortia were lower than that in the Z. mobilis 8b mono-fermentation. It was shown that, in general, the higher the ratio of Z. mobilis 8b applied, the higher the rates of xylose assimilation achieved. In special, when S. stipitis CICC1960 : Z. mobilis 8b = 3:1 (initial inoculum size proportion), the xylose assimilation rate was 0.92 g/L/h; when S. stipitis CICC1960 : Z. mobilis 8b = 1:3, the xylose assimilation rate improved to 2.38 g/L/h. At 21 h, the consortia fermentation reached the endpoint of fermentation. Interestingly, when S. stipitis CICC1960 : Z. mobilis 8b = 1:3, the xylose consumption reached 36.73 g/L, which was signi cantly (P < 0.01) higher than that in the S. stipitis CICC1960 mono-fermentation and that in the Z. mobilis 8b mono-fermentation (Additional le 1: Table S1). Correspondingly, the ethanol titer of this consortium reached 57.21 g/L, which was 48.37% higher than that of the S. stipitis CICC1960 mono-fermentation and 6.52% higher than that of the Z. mobilis 8b mono-fermentation. These results (S. stipitis CICC1960 : Z. mobilis 8b = 1:3) were comparable with or better than other consortia fermentations listed in Table 1, in terms of xylose removal e ciency, ethanol yield, and ethanol productivity. 2.2 Genetic engineering of Z. mobilis 8b In Z. mobilis, ZMO0256 encoding D-lactate dehydrogenase is involved in the production of lactate as a byproduct; ZMO0689 encoding glucose-fructose oxidoreductase participates in xylitol and sorbitol production (23). It was demonstrated that disruption of ZMO0689 could improve xylose fermentation performance of Z. mobilis (24). In order to introduce one copy of xylose metabolic genes (xylA, xylB, tktA, talB) into the Z. mobilis 8b genome and improve its xylose assimilation performance, the engineered strains Z. mobilis FR1 (ZMO0256::P pdc -talB-tktA) and Z. mobilis FR2 (ZMO0256::P pdc -talB-tktA; ZMO0689::P pdc -xylA-xylB) were sequentially constructed.
The fermentation performances of Z. mobilis FR1 and Z. mobilis FR2 were evaluated in 80G40XRM and were compared with their parental stain Z. mobilis 8b. As shown in Fig. 2a, the three strains did not differ in their glucose assimilation pro les, and glucose was depleted within 8.5 h. For the xylose consumption and ethanol production pro les ( Fig. 2b and 2c), Z. mobilis FR1 did not show much difference with Z. mobilis 8b. However, Z. mobilis FR2 accelerated its xylose assimilation rate in the mid-to-late fermentation period (Fig. 2b): the xylose consumption and ethanol production achieved by Z. mobilis FR2 were increased by 15.36% and 6.81% respectively at 20.5 h as compared with Z. mobilis 8b (Additional le 1: Table S2). Besides, the ethanol productivity of Z. mobilis FR2 was 5.08 g/L/h, which was signi cantly (P < 0.01) higher than Z. mobilis 8b (4.84 g/L/h). The ethanol yield of Z. mobilis FR2 was 95.47%, which was comparable with A3 (96.6%) and AD50 (96%), the two best strains developed so far by adaptive laboratory evolution (23,25).
However, as shown in Fig. 2a and 2b, though CCR was alleviated in Z. mobilis FR2 fermentation, its xylose utilization rate was still lower than its glucose utilization rate, and there was still a slight amount of xylose that remained (approximately 2.5 g/L) at the endpoint. These results agreed with other Z. mobilis strains, including C25, 39676/pZB4L (26), ZM4/Ac R (pZB5, pJX1) (27), A3 (23), and AD50 (25), yet no exact reason of the incomplete xylose utilization has been identi ed thus far. In Z. mobilis, xylose is transported through a glucose facilitated diffusion protein (25), which is a native glucose transporter and has low a nity to xylose. This low a nity xylose transport might be the burden behind the abovementioned problems. Further investigation into this eld would greatly promote the commercialization of cellulosic ethanol.

Effect of oxygen on Z. mobilis FR2 fermentation
In the above experiments, as S. stipitis could not grow under static cultivation, 150 rpm was applied in the S. stipitis mono-fermentation, the Z. mobilis mono-fermentation, and the consortia fermentation that consisted of the two species. However, as Z. mobilis is a facultative anaerobe, it can ferment under both static and aerobic conditions. Therefore, a further study was conducted to check whether there was any difference in Z. mobilis FR2 fermentation pro les under static and agitated (150 rpm) conditions in 80G40XRM. As shown in Fig. 3, oxygen signi cantly boosted Z. mobilis FR2's glucose (P < 0.05) and xylose (P < 0.01) consumption rates and improved ethanol productivity by 54.51% (P < 0.01). However, at fermentation endpoint (27 h), the static fermentation of Z. mobilis FR2 showed an increase in xylose consumption and ethanol production by 1.65 g/L and 3.45 g/L, respectively compared with that in agitated fermentation. To achieve high ethanol productivity, ethanol fermentation under 150 rpm was kept in this study.
In theory, the 1.65 g/L more xylose consumed by Z. mobilis FR2 in static condition should be transformed into 0.84 g/L ethanol (the theoretical ethanol yield is 0.51 g/g xylose). However, the real difference in ethanol titers between the static fermentation and agitated fermentation was as high as 3.45 g/L. This suggests oxygen had a negative effect on ethanol production by Z. mobilis FR2. In 1990, Tanaka et al. found that Z. mobilis produced a little more acetaldehyde (0.28-4.49 g/L) when oxygen was supplied (28). Acetaldehyde was primarily produced by NADH dehydrogenase, one of the key components in the Z. mobilis respiratory chain. This enzyme has the same cofactor (NADH) as ethanol dehydrogenase. During aerobic fermentation, the activity of NADH dehydrogenase was higher than that of ethanol dehydrogenase. Therefore, a large amount of NADH was used to reduce the dissolved oxygen concentration in the medium. Due to the lack of su cient NADH, the transformation from acetaldehyde to ethanol by ethanol dehydrogenase was inhibited, and thus negatively affected the ethanol production of Z. mobilis under agitated cultivation (29). One evidence for this hypothesis is that for Z. mobilis mutant strains, whose NADH dehydrogenase is defective, this negative effect of oxygen on Z. mobilis ethanol fermentation could be alleviated (30-32).

Assimilation of corn stover hydrolysates by consortium composed of S. stipitis CICC1960 and Z. mobilis FR2
First, it was evaluated whether Z. mobilis FR2 could outcompete Z. mobilis 8b in corn stover hydrolysate fermentation. As shown in Fig. 4a, a difference was not observed regarding glucose assimilation ability of the two strains: glucose assimilation rates were 2.54 g/L/h and 2.51 g/L/h for Z. mobilis FR2 and Z. mobilis 8b, respectively, and glucose was depleted within 30 h. As for xylose assimilation (Fig. 4b), though the two strains did not exhibit a difference during the rst 24 hours, the assimilation rate of Z. mobilis 8b gradually decreased, while the rate for Z. mobilis FR2 remained stable. At 60 h, Z. mobilis FR2 assimilated 14.60 g/L xylose, which was signi cantly higher (P < 0.05) than that of Z. mobilis 8b (10.60 g/L). Additionally, Z. mobilis FR2 produced 37.13 g/L ethanol in 120 g/L corn stover hydrolysate (concentration here refers to the total amount of glucose and xylose in corn stover hydrolysate before sterilization), while Z. mobilis 8b produced 34.93 g/L ethanol (Fig. 4c). These results agreed with the fermentation results in 80G40XRM (Fig. 2). Due to the better xylose assimilation ability of Z. mobilis FR2, Z. mobilis FR2 was used to replace Z. mobilis 8b in the next consortium fermentation with S. stipitis CICC1960 in corn stover hydrolysates.
As shown in Fig. 5, while consortium fermentation (S. stipitis CICC1960 : Z. mobilis FR2 = 1:3) and Z. mobilis FR2 mono-fermentation did not show any difference in glucose and xylose consumption rates and amounts in the 60 and 90 g/L corn stover hydrolysates fermentation, the consortium produced slightly more ethanol (~ 0.86 g/L) than the Z. mobilis FR2 mono-fermentation (Table 2). Additionally, the consortium fermentation in the two cases was better than the S. stipitis CICC1960 mono-fermentation in terms of glucose assimilation, xylose assimilation, and ethanol production rates and quantities, and did not exhibit strong CCR which was evident in the S. stipitis CICC1960 mono-fermentation (Fig. 5). For the 120 g/L corn stover hydrolysate fermentation, the glucose assimilation rate of the consortium (2.83 g/L/h) was slightly slower than Z. mobilis FR2 (3.24 g/L/h), while xylose assimilation rates were nearly the same prior to 36 h. However, the consortium nally produced 33.05 g/L ethanol at endpoint, which was 1.02 g/L higher than the Z. mobilis FR2 mono-fermentation and 16.7 g/L higher than the S. stipitis CICC1960 mono-fermentation (Table 2).  (17). Though the productivity in their study was high (1.868 g/L/h), the actual sugar concentration in the hydrolysate was only 11 g/L glucose, 4 g/L xylose, and 4 g/L cellobiose, which is impractical in real applications. These comparisons demonstrated that the consortium fermentation mode utilized in this study has an edge in ethanol production compared with other existed modes of S. stipitis and Z. mobilis co-fermentation.
We note that the ethanol titer and productivity values in the 120 g/L corn stover hydrolysate were lower than the values in 80G40XRM ( Table 2, Additional le 1: Tables S1 and S2). This was because many of the inhibitors, like phenols, were presented in the corn stover hydrolysate (33). These inhibitors negatively affected the microbial tness in the lignocellulosic hydrolysate and thus reduced the ethanol yield and productivity. Although it has been shown that the immobilization of microbes could alleviate the negative effect to some degree, the exact mechanisms are not clear (34).
Duong Thi Thuy Nguyen et al. found that the presence of living Z. mobilis cells negatively affected the xylose assimilation performance of S. stipitis, suggesting that there might be an amensalism relationship between the two species (19,35). Similarly, in our study, when the initial inoculum OD 600 was controlled to 0.1 in the 80G40XRM fermentation, no improvement was observed in the consortium fermentation pro les compared with the Z. mobilis mono-fermentation pro les (data not shown). In contrast, when a high inoculum size (an initial OD 600 of approximately 1.8) was used, the consortium of S. stipitis and Z.
mobilis assimilated more xylose and produced more ethanol in both the 80G40XRM and 120 g/L corn stover hydrolysate. This implied a commensalism or cooperation relationship between the two species during fermentation (35). Perhaps this positive relationship could only be exhibited under speci c conditions, such as under a high cell density.
Exploring the interactions between S. stipitis and Z. mobilis under various cell densities, as well as the ethanol fermentation performance in corn stover hydrolysate by co-immobilized cells of the two species, are our future research goals. Indeed, a deep understanding of the interactions between S. stipitis and Z. mobilis and the mechanisms behind the protective role of co-immobilization on cells will pave the way to further enhance the consortium fermentation performance while lowering the inoculum size, and will help promoting the industrial progression of cellulosic ethanol production.

Conclusions
This study focused on evaluating a fermentation method to e ciently transform glucose and xylose to ethanol via arti cial consortia composed of suspended S. stipitis and suspended Z. mobilis. By fermentation process optimization and genetic engineering, the consortium built here exhibited enhanced xylose assimilation ability and ethanol production performance in both 80G40XRM and corn stover hydrolysate, and did not experience evident CCR phenomenon. Hence, this study proved that S. stipitis and Z. mobilis could be co-cultured together in suspension in cellulosic ethanol production and provided a novel strategy for further application.

Strains, plasmids and primers
S. stipitis CICC1960 was purchased from the China Center of Industrial Culture Collection. Z. mobilis 8b was kindly given by Shihui Yang, Hubei University, and was used as the starting strain for further strains constructions. Plasmid Pmini was kindly given by Nan Peng, Huazhong Agricultural University. All primers were synthesized by Tsingke Biotechnology Co., Ltd. (Chengdu, China) and puri ed via polyacrylamide gel electrophoresis.
All strains, plasmids and sgRNAs are listed in Additional le 1: Table S3. Primers are listed in Additional le 1: Table S4.
The treatment parameters were 140°C and 6 h.
The pretreated corn stover was washed intensively with water or diluted HNO 3 until the pH of the washing water turned neutral. The washed corn stover was oven dried and stored in sealed bags at room temperature.

Enzymatic hydrolysis
Pretreated corn stover (10 g) was mixed with 100 mL citric acid buffer (8.823 g/L tri-sodium citrate dihydrate, 3.843 g/L citric acid, pH 4.8) and 5 mL cellulase (Sigma-Aldrich, Saint Louis, US). Afterwards, the mixture was placed in an incubator at 50°C and 150 rpm for 72 h. When the enzymatic hydrolysis was nished, the hydrolysate was centrifuged twice at 3000 g for 25 min each time. The hydrolysate was then centrifuged again at 10000 g for 5 min for the further removal of the remaining solids. Sugars in the lignocellulosic hydrolysate were concentrated using a rotary evaporator to attain 60 g/L, 90 g/L, and 120 g/L hydrolysates with regard to the concentrations of total glucose and xylose. Subsequently, 10 g/L (v/v, the ratio of seed culture volume/ fermentation culture volume).
The total fermentation volume was 50 mL for 80G40XRM fermentation and 10 mL for corn stover hydrolysates fermentation. The fermentation conditions were 30°C and 150 rpm, unless there was further indication in the portion for exploring the effect of oxygen on Z. mobilis fermentation (as seen in 2.3).
Four replicates were performed for the 80G40XRM fermentation, while three replicates were performed for the corn stover hydrolysates fermentation.

Determination of glucose, xylose and ethanol concentrations during fermentation
The glucose, xylose, and ethanol titers in the fermentation samples were analyzed by an Agilent 1200 Series HPLC system (Agilent Technologies, Santa Clara, US) equipped with a Bio-Rad HPX-87H column (Bio-Rad Laboratories, Richmond, US). The mobile phase was 5 mM H 2 SO 4 . The operating parameters were 20 µL injection volume, 0.6 mL/min rate, and 35°C.

Calculation of ethanol productivity and yield
Ethanol productivity = Ethanol titer / fermentation time Ethanol yield = Ethanol titer / glucose and xylose consumed The theoretical ethanol yield is 0.51 g/g sugars consumed.

Statistical analysis
Data are presented as mean ± standard error. All gures were prepared using Prism 8 (GraphPad Software, LLC).
Signi cant differences were statistically analyzed using IBM® SPSS® Statistics (Version 22, US). If P > 0.05 in the homogeneity of variance test, a one-way ANOVA followed by Turkey test was used. Otherwise, a nonparametric test (Kruskal-Wallis H) was used.

Declarations
Ethics approval and consent to participate Not applicable.

Consent for publication
Not applicable.
Availability of data and materials Not applicable. Figure 1 Fermentation pro les of consortia consisting of S. stipitis CICC1960 and Z. mobilis 8b in 80G40XRM. a Glucose assimilation pro les. b Xylose assimilation pro les. c Ethanol production pro les. Data are mean ± standard error from four replicates Figure 2 Fermentation pro les of Z. mobilis 8b, Z. mobilis FR1, and Z. mobilis FR2 in 80G40XRM. a Glucose assimilation pro les. b Xylose assimilation pro les. c Ethanol production pro les. Data are mean ± standard error from four replicates Figure 3 Fermentation pro les of Z. mobilis FR2 in 80G40XRM with different rotation speeds. a Glucose assimilation pro les. b Xylose assimilation pro les. c Ethanol production pro les. Data are mean ± standard error from four replicates Fermentation pro les of Z. mobilis 8b and Z. mobilis FR2 in corn stover hydrolysate. a Glucose assimilation pro les. b Xylose assimilation pro les. c Ethanol production pro les. The total concentrations of glucose and xylose in the hydrolysate was 120 g/L before autoclave sterilization. Data are mean ± standard error from three replicates Consortium fermentation pro les in various concentrations of corn stover hydrolysates. a, b, c Glucose, xylose and ethanol fermentation pro les in 60 g/L of corn stover hydrolysate. d, e, f Glucose, xylose and ethanol fermentation pro les in 90 g/L of corn stover hydrolysate. g, h, i Glucose, xylose and ethanol fermentation pro les in 120 g/L of corn stover hydrolysate. The concentration of corn stover hydrolysates represents the total amount of glucose and xylose in the hydrolysates before autoclave sterilization. For consortium fermentation, the inoculation ratio between S. stipitis CICC1960 and Z. mobilis FR2 was 1:3.

Figures
Data are mean ± standard error from three replicates