Simultaneous Fermentation of Glucose and Xylose to Butanol by Clostridium sp. Strain BOH3

Abstract

Cellulose and hemicellulose constitute the major components in sustainable feedstocks which could be used as substrates for biofuel generation. However, following hydrolysis to monomer sugars, the solventogenic Clostridium will preferentially consume glucose due to transcriptional repression of xylose utilization genes. This is one of the major barriers in optimizing lignocellulosic hydrolysates that produce butanol. Unlike studies on existing bacteria, this study demonstrates that newly reported Clostridium sp. strain BOH3 is capable of fermenting 60 g/liter of xylose to 14.9 g/liter butanol, which is similar to the 14.5 g/liter butanol produced from 60 g/liter of glucose. More importantly, strain BOH3 consumes glucose and xylose simultaneously, which is shown by its capability for generating 11.7 g/liter butanol from a horticultural waste cellulosic hydrolysate containing 39.8 g/liter glucose and 20.5 g/liter xylose, as well as producing 11.9 g/liter butanol from another horticultural waste hemicellulosic hydrolysate containing 58.3 g/liter xylose and 5.9 g/liter glucose. The high-xylose-utilization capability of strain BOH3 is attributed to its high xylose-isomerase (0.97 U/mg protein) and xylulokinase (1.16 U/mg protein) activities compared to the low-xylose-utilizing solventogenic strains, such as Clostridium sp. strain G117. Interestingly, strain BOH3 was also found to produce riboflavin at 110.5 mg/liter from xylose and 76.8 mg/liter from glucose during the fermentation process. In summary, Clostridium sp. strain BOH3 is an attractive candidate for application in efficiently converting lignocellulosic hydrolysates to biofuels and other value-added products, such as riboflavin.

INTRODUCTION

Conversion of lignocellulosic biomass to biofuels (e.g., butanol) is usually limited by the inefficiency of the bacteria in the utilization of pentoses, which constitute 20 to 60% of the sugars in the hydrolysate of lignocellulose (39). So far, the biofuel-generating microbes in common use are either unable to utilize pentoses at all (e.g., Saccharomyces cerevisiae) or consume hexoses first and then pentoses with a low yield. Good examples are the solventogenic Clostridium species (e.g., C. acetobutylicum, C. beijerinckii, and C. pasteurianum), which are among the few microorganisms able to ferment both pentose and hexose sugars (10). However, the solvents, including butanol, are still produced mainly from glucose, while small amounts are produced from xylose, as observed in cultures of C. acetobutylicum ATCC 824 and C. beijerinckii NCIMB 8052 when fermenting a mixture of glucose and xylose (14, 17, 38). Gu et al. have shown that C. acetobutylicum ATCC 824 utilized 86% of glucose within 40 h, whereas only 6% of xylose was consumed even after an elongated incubation time (14). This sequential utilization extends fermentation time and results in incomplete substrate consumption (21, 33). The phenomenon of preferring glucose over xylose is referred to as carbon catabolite repression, in which microorganisms preferentially utilize a rapidly metabolizable carbon source and inhibit the expression of some genes and enzyme activities related to the catabolism of nonpreferred carbon resources (21, 33). Unless glucose and xylose are both utilized efficiently, converting lignocellulosic biomass into biologically based products is unfavorable from an economic viewpoint (19, 20, 22), as yields would be limited. Hence, in order to fully utilize the sugars in the hydrolysates of lignocellulosic biomass, microbes capable of fermenting both glucose and xylose with a high butanol yield are highly desirable to improve the efficiency of biofuel production systems.

In addition to butanol production, certain biofuel-generating microbes, such as Clostridium, can produce other value-added products (e.g., riboflavin, also known as vitamin B2), which adds to the value of the butanol fermentation process. Actually, in addition to producing butanol, Clostridium species are also one of the commercial microorganisms used to produce riboflavin on an industrial scale, although they only generate a very small amount of riboflavin (<50 mg/liter) (16, 26, 27). Riboflavin, a yellow, water-soluble vitamin, is biosynthesized solely by plants and numerous microorganisms but not by vertebrates. Thus, this vitamin has to be supplied by foods and dietary supplements. Currently, industrial production has shifted completely from chemical synthesis to microbial fermentation (32), which has become one of the major fermentation products in the biotechnology industry, with an annual market demand of several million kilograms (31). Therefore, the coproduction of butanol and riboflavin would make the fermentation process more economically feasible (28).

The aim of this study is to explore wild-type Clostridium sp. strain BOH's capability to utilize the five-carbon sugar xylose. A further study then was conducted on both butanol and riboflavin production from fermenting a mixture of glucose and xylose and hydrolysates from horticultural wastes. Lastly, the activities of xylose-utilizing enzymes (xylose isomerase and xylulose kinase) in strain BOH3 were evaluated to determine the underlying mechanism of cofermentation of glucose and xylose.

MATERIALS AND METHODS

Growth medium and culture conditions.

Clostridium sp. strain BOH3 and Clostridium sp. strain G117 were used in this study (6, 9). Batch cultures were grown at 35°C in defined mineral salts medium containing (per liter of distilled water) the following: KH₂PO₄, 0.75 g; K₂HPO₄, 0.75 g; CH₃COONH₄, 2 g; and yeast extract, 5 g. In addition, 1 ml of trace element solution (35), 1 ml of Na₂SeO₃-Na₂WO₄ solution (7), and 10 mg of resazurin were added to 1 liter of the medium. After the medium was boiled and cooled to room temperature under N₂, reductants Na₂S, l-cysteine, and dl-dithiothreitol were added to a final concentration of 0.2, 0.2, and 0.5 mM, respectively (15). Subsequently, 20 mM 2-(N-morpholino) ethanesulfonic acid (MES) was added to the medium to adjust its initial pH to 6.0. The medium then was dispensed to serum bottles, which were sealed with butyl stoppers, autoclaved for 20 min, and cooled to room temperature. Glucose, xylose, or a mixture of glucose and xylose was amended to the medium described above before inoculation.

Cultures for inoculation were grown in 50 ml mineral salts medium amended with glucose, xylose, or a glucose-xylose mixture at 35°C for ∼20 h (late exponential phase) unless otherwise stated. Inocula of 5 ml were added to 45 ml of the reduced mineral salts medium in 160-ml serum bottles, which were incubated in a shaker at a rotary rate of 150 rpm at 35°C. The pH was adjusted to 5.0 using 2 M NaOH after 24 h. Experiments were carried out in duplicate.

Preparation of horticultural waste hydrolysates.

Horticultural wastes collected from a horticultural waste treatment plant (ecoWise Solution Pte. Ltd.) were used as raw materials and were dried and milled to a small size (200 to 500 μm). Pretreatment of the horticultural waste was carried out by using the organosolv method as described by Zhang et al. (40) and Geng et al. (12). Initially, the organic solvent ethanol was used to dissolve hemicellulose and lignin to the solvent phase. The ethanol then was removed by vacuum evaporation, and the hemicellulosic fraction carrying mainly xylose was further detoxified using activated charcoal powder and concentrated to obtain a final sugar mixture containing 5.9 g/liter of glucose and 58.3 g/liter of xylose (40). The solid residues from the pretreatment step contained mainly cellulose, which was hydrolyzed by enzymes of Celluclast 1.5L and Novozyme 188 to glucose and designated cellulosic hydrolysate (12). Supplemental xylose (16.9 g/liter) was added to the cellulosic hydrolysate to attain a 2:1 ratio of glucose (39.8 g/liter) to xylose (20.8 g/liter). Prior to filter sterilization, the pHs of the detoxified hemicellulosic hydrolysate (containing 5.9 g/liter of glucose and 58.3 g/liter of xylose) and cellulosic hydrolysate (containing 39.8 g/liter of glucose and 20.8 g/liter of xylose) were adjusted to 6.0 by titrating with 10 M NaOH solution. This was followed by supplementing KH₂PO₄, K₂HPO₄, CH₃COONH₄, yeast extract, trace element solution, Na₂SeO₃-Na₂WO₄ solution, and reductants as described in the above section.

Expression of genes related to xylose utilization as measured by qPCR.

Total RNA from cell pellets of Clostridium sp. strain BOH3 and G117 was extracted by using a modified TRIzol method and RNeasy minikit (Qiagen, Hilden, Germany) according to the supplier's instructions. The remaining trace DNA was removed using an RNase-free DNase kit (Qiagen, Hilden, Germany). cDNA was synthesized in one step using an iScript One-Step reverse transcription-PCR (RT-PCR) kit (Bio-Rad Laboratories, Munich, Germany). A quantitative real-time PCR (qPCR) (ABI 7500 Fast real-time PCR system; ABI, Foster City, CA) assay was performed by using QuantiTest SYBR green (Qiagen, GmBH, Germany) to quantify the transcription levels of the xylose isomerase gene. The xylose isomerase gene-specific primers used for qPCR were designed based on the xylose isomerase gene sequence of cultured BOH3 and G117, with the forward primers 5′-ATGTTGCAGTTACAGAGGGAGA-3′ (strain BOH3) and 5′-CTGTTTTACTAATCCAA GGTATGTTCA-3 (strain G117) and reverse primers 5′-TTTCATCTTGGCTTACCTT GTC-3′ (strain BOH3) and 5′-ATCTAGCTCTTTTGTTATTTCA ATTGC-3′ (strain G117). Primers for the housekeeping genes [(3R)-hydroxymyristoyl-ACP dehydratase (fabZ) for strain BOH3 (23) and peptidase T (pepT) for strain G117 (36)] are the following: strain BOH3-forward, 5′-AAATAGAACCAGGGAAAAGAGCA-3′; strain BOH3-reverse, 5′-GCAACACCACCAAGTTGAGC-3′; strain G117-forward, 5′-TGATGGAG GCGAGGAAGGTG-3′; and strain G117-reverse, 5′-CATTGTATTCTTTGCAGACCCTGG-3′. The following real-time PCR program was used: 10 min at 95°C, 45 cycles of 10 s at 95°C, and 30 s at 55°C, followed by a melting-point analysis (45 to 95°C) after the final PCR step to verify the specificity of amplified products. The real-time PCR data were analyzed with iQ5 software, and relative quantification was done by using the ΔΔC_T method (25) and normalized to the abundance of housekeeping genes.

Xylose isomerase and xylulose kinase assays.

Cell extracts obtained from the batch cultures were used for enzymatic activity assays. The chemicals used in the enzymatic activity assessment were obtained from Sigma-Aldrich (St. Louis, MO). Cultures at the exponential phase were harvested by centrifugation at 10,000 × g at 4°C for 10 min. Cell pellets were washed once with extraction buffer (100 mM Tris-HCl [pH 7.5], 20 mM KCl, 20 mM MgCl₂, 5 mM MnSO₄, 0.1 mM EDTA, and 2 mM _DL-dithiothreitol). The resulting cell pellets were sonicated using an ultrasonic homogenizer in an ice water bath for 2 min followed by 2-min cooling intervals, which were repeated three times. Cell debris was removed by ultracentrifugation (12,000 × g, 4°C, 30 min). The cell lysates were used subsequently for enzyme assays.

The activity of xylose isomerase of the whole-cell lysate was determined in a 1-ml reaction volume containing 70 mM xylose, 20 mM MgCl₂, 5 mM MnSO₄, and 2 mM _DL-dithiothreitol in 100 mM Tris buffer (pH 7.5) (30). The reaction mixture was incubated for 30 min at 30°C, and then 0.5 M HClO₄ was added to stop the reaction. The xylulose produced was quantified by using the cysteine-carbazole-sulfuric acid assay by measuring the absorbance at 540 nm (11). The enzyme activity of xylulose kinase was determined by the reduction of xylulose in the reaction mixture as described previously, with minor modifications (30). The reaction mixture contained the following: 50 mM Tris-HCl buffer (pH 7.5), 2.0 mM MgCl₂, 2.0 mM ATP, 0.2 mM phosphoenolpyruvate, 0.2 mM NADH, 10 U pyruvate kinase (EC 2.7.1.40), 10 U lactate dehydrogenase (EC 1.1.1.27), and 8.5 mM xylulose. Consumption of NADH was measured by a spectrophotometer at 340 nm. One unit of enzyme activity was defined as the amount of enzyme which generated 1 μmol of product per min. The protein amount in the cell extracts was detected by using the Coomassie protein assay reagent (5).

Analytical methods.

The biomass in the fermentation broth was determined on a UV-visible spectrophotometer set at a wavelength of 600 nm (Lambda-25; Perkin-Elmer, USA). Riboflavin initially was identified and quantified by using high-performance liquid chromatography (HPLC) and later was quantified at a wavelength of 444 nm on a Beckman DU-800 spectrophotometer based on a modified protocol from Sauer et al. (29). Briefly, the fermentation broth was diluted with 0.1 M phosphate buffer and heated at 80°C for 10 min before centrifugation. The supernatant then was assayed. Similarly, for sugar and biosolvent analysis, culture broths were centrifuged at 10,000 × g for 10 min at 4°C, and the supernatant fluids were stored at −20°C until further analysis. The concentrations of glucose and xylose were measured using the YSI Life Sciences 7100 MBS multiparameter biochemistry analyzer. Glucose and xylose sensors are configured in the same electrode chamber, allowing simultaneous measurement of these sugars in 1 min. Biosolvents (i.e., acetone, ethanol, and butanol) were measured by a gas chromatograph (GC; model 7890A; Agilent Technologies, USA) on a Durabond (DB)-WAXetr column (30 m by 0.25 mm by 0.25 μm; J&W, USA) equipped with a flame ionization detector (FID). The oven temperature was held initially at 60°C for 2 min, increased at 15°C/min to 230°C, and then held for 1.7 min. Helium was used as the carrier gas, with a column flow rate of 1.5 ml/min. Five-point standard curves were established by running standard solutions containing acetone, butanol, and ethanol.

RESULTS

Efficient coproduction of butanol and riboflavin from xylose by strain BOH3.

In contrast to the two most well-known butanol-producing species, C. acetobutylicum ATCC 824 and C. beijerinckii NCIMB 8052, the newly discovered Clostridium sp. strain BOH3 could produce slightly larger amounts of butanol (7.4 versus 7.0 g/liter) from xylose than from glucose (30 g/liter each) (6). This observation inspired further investigation on strain BOH3's capability to utilize xylose at a higher concentration. When 60 g/liter of xylose was fed as the sole carbon source to cultures of BOH3, cells multiplied rapidly (the optical density at a wavelength of 600 nm reached 4.9) during the first 28 h with only small amounts of butanol production (3.4 g/liter). After that, acetone, butanol, and ethanol concentrations increased rapidly to highest values of 5.3, 14.9, and 1.2 g/liter, respectively, within 62 h (Fig. 1A and B). This corresponds to a yield of 0.25 g/g for butanol and 0.36 g/g for total solvents of acetone, butanol, and ethanol (ABE). Interestingly, riboflavin also appeared during the fermentation process and reached a concentration of 110.5 mg/liter, which is much higher than those found in previous studies using either wild-type (50 mg/liter) or gene-modified Clostridium strains (70 mg/liter) (3, 8). In comparison, when glucose (60 g/liter) was used as the sole carbon source, the substrate consumption curves almost overlapped with that of xylose (Fig. 1A and C). Strain BOH3 exhibited a similar sugar consumption rate of 0.83 g/liter/h and generated slightly smaller amounts of riboflavin (76.8 mg/liter), butanol (14.5 g/liter), and ABE (20.1 g/liter) than those using xylose as a sole substrate (Fig. 1C and D). Thus, strain BOH3 represents the first wild-type solventogenic Clostridium that could ferment xylose to butanol (14.9 g/liter) more efficiently than previously reported wild-type (5.8 g/liter) or gene-modified (11.6 g/liter) Clostridium strains under similar operational conditions (Table 1).

FIG 1.

Growth and fermentation profiles of Clostridium sp. strain BOH3 in reduced mineral medium containing 60 g/liter of xylose (A and B) and 60 g/liter of glucose in batch bottles (C and D).

TABLE 1.

Xylose consumption and total solvent (acetone, butanol, and ethanol) production by different Clostridium species fed with 60 g/liter xylose

Strain	Gene type	Xylose concn (g/liter)		Solvent titer (g/liter)		Reference or source
		Initial	Residual	Butanol	ABE
C. acetobutylicum ATCC 824	Wild type	60	36.7 ± 0.2	4.2 ± 0.1	6.7 ± 0.2	14
C. acetobutylicum 824-TALa	Modified	60	32.5 ± 0.3	5.1 ± 0.2	8.1 ± 0.2	14
C. acetobutylicum ATCC 824	Wild type	60	35.7 ± 0.5	4.3 ± 0.3	6.8 ± 0.1	17
C. acetobutylicum EA2018b	Mutant	60	23.6 ± 1.2	8.5 ± 0.1	11.6 ± 0.1	17
C. beijerinckii NCIMB 8052	Wild type	60	27.3 ± 0.9	5.8 ± 0.2	7.9 ± 0.7	38
C. beijerinckii 8052xylR-xylTptbc	Modified	60	4.2 ± 0.1	11.6 ± 0.1	15.9 ± 0.3	38
Clostridium sp. strain G117	Wild type	60	24.6 ± 0.6	5.2 ± 0.1	7.2 ± 0.1	This study
Clostridium sp. strain BOH3	Wild type	60	0	14.9 ± 0.2	21.4 ± 0.2	This study

^aC. acetobutylicum 824-TAL is a transformant from C. acetobutylicum ATCC 824 bearing the E. coli transaldolase (tal) gene (14).

^bC. acetobutylicum EA2018 is a mutant obtained from C. acetobutylicum ATCC 824 after several rounds of mutagenesis using NTG (N-methyl-N′-nitrosoguanidine) (17).

^cC. beijerinckii 8052xylR-xylT_ptb is an engineered strain from C. beijerinckii NCIMB 8052 (putative xylose repressor gene [xylR] inactivation plus xylose proton-symporter gene [xylT] overexpression driven by the ptb promoter) (38).

Simultaneous utilization of glucose and xylose by strain BOH3.

Glucose and xylose are the two major components in the hydrolysates obtained after pretreatment of lignocelluloses and enzymatic saccharification. As the ratio of glucose and xylose varies widely among different lignocellulosic biomass types, strain BOH3 was fed with a mixture of glucose and xylose at different ratios (∼2:1, 1:1, and 1:2) (Fig. 2). In any ratio of glucose-xylose mixture, strain BOH3 rapidly and simultaneously fermented both glucose and xylose to produce butanol, acetone, ethanol, and riboflavin (Fig. 2). In contrast, previous studies exhibited typical sequential utilization, consuming glucose first and then xylose (9, 14, 24, 36, 37, 38). For instance, when providing a mixture of glucose and xylose (30 g/liter each), strain G117 consumed all of the glucose in the first 48 h, and xylose utilization was initiated only after the glucose was completely consumed (after 48 h), resulting in a total of 6.5 g/liter butanol after 100 h of fermentation. In comparison, strain BOH3 consumed all of the glucose and most of the xylose in the glucose-xylose mixtures after 72 h of incubation (Fig. 2A, C, and E). More xylose in the glucose-xylose mixture led to slightly larger amounts of butanol and riboflavin being produced (Fig. 2B, D, and F). For example, when fed with glucose and xylose in a ratio of 1:2, strain BOH3 produced the highest levels of butanol (13.0 g/liter) and riboflavin (91.4 mg/liter) among the above-described three substrate ratios, which is consistent with the results in Fig. 1 showing higher butanol and riboflavin production after consuming the same amount of xylose as glucose.

FIG 2.

Growth and fermentation profiles of Clostridium sp. strain BOH3 in reduced mineral medium containing a mixture of 30 g/liter of glucose and 30 g/liter of xylose (A and B), 40 g/liter of glucose and 20 g/liter of xylose (C and D), and 20 g/liter g of glucose and 40 g/liter of xylose (E and F).

To further investigate the variation between glucose and xylose utilization, the culture initiated with glucose or xylose as the sole carbon source was supplemented with xylose or glucose after 24 h of incubation (Fig. 3). For the initial 24-h cultivation on 30 g/liter glucose, the average glucose consumption rate was 0.54 g/liter/h. Xylose then was introduced to a final concentration of 30 g/liter at the 24-h time point (while 17 g/liter of glucose remained). During the next 20 h (the period from 24 to 44 h), xylose was consumed at an average rate of 0.33 g/liter/h, whereas the glucose consumption rate decreased to 0.23 g/liter/h (Fig. 3A). However, the total sugar consumption rate was 0.56 g/liter/h, which was nearly equal to the rate before xylose supplementation. Similar results were obtained when glucose was introduced after 24 h to the culture initiated with xylose (Fig. 3B); glucose was consumed at 0.33 g/liter/h, and the xylose consumption rate was reduced from 0.57 g/liter/h to 0.22 g/liter/h. The total sugar consumption rate of 0.55 g/liter/h was very close to the rate before glucose supplementation. Therefore, when the medium was supplemented with a second sugar, the sugar consumption capacity was redistributed between the two sugars.

FIG 3.

Sugar consumption profiles. After 24 h of cultivation, xylose was added to glucose-containing medium (A), and glucose was added to xylose-containing medium (B).

Fermentation of lignocellulosic hydrolysate by strain BOH3.

To further test strain BOH3's capability to ferment both glucose and xylose in the hydrolysate of lignocellulosic biomass, horticultural waste was chosen for the subsequent experiment. After pretreatment, enzymatic hydrolysis, and xylose supplementation (12), the horticultural cellulosic hydrolysate contained 39.8 g/liter glucose and 20.5 g/liter xylose in a ratio of ∼2:1. Strain BOH3 fermented both glucose and xylose in the hydrolysate simultaneously after an adaption phase of 28 h (Fig. 4A). With a glucose/xylose ratio of 2:1, strain BOH3 consumes glucose and xylose at the same rate whether the sugars come from hydrolysate or pure chemicals (Fig. 3A versus Fig. 2C). However, strain BOH3 produced 11.2 g/liter butanol from the hydrolysate medium and had a slightly lower yield, 0.19 g/liter, than the glucose-xylose mixture medium (12.4 g/liter butanol). Riboflavin was also detected at a concentration of 51.2 mg/liter in the hydrolysate-spiked medium (Fig. 4A), which is lower than the level in the glucose-xylose mixture medium (91.4 mg/liter) (Fig. 2F). On the other hand, the hemicellulosic hydrolysate obtained from the horticultural waste was further concentrated and detoxified, resulting in a content mainly of xylose (58.3 g/liter) and a small amount of glucose (5.9 g/liter) (Fig. 4B) (40). After a 28-h lag, strain BOH3 rapidly converted all of the xylose into 65.3 mg/liter riboflavin and 11.9 g/liter butanol with a final yield of 0.19 g/g, which is similar to fermenting the mixture of glucose-xylose (Fig. 2). Therefore, strain BOH3 is able to simultaneously ferment both glucose and xylose in hydrolysates of lignocellulosic biomass.

FIG 4.

Fermentation of cellulosic hydrolysate (containing 39.8 g/liter glucose and 20.8 g/liter xylose) from horticultural waste (A) and hemicellulosic hydrolysate (containing 5.9 g/liter glucose and 58.3 g/liter xylose) from horticultural waste (B) by Clostridium sp. strain BOH3.

Expression of xylose isomerase and xylulokinase in strain BOH3.

Xylose isomerase (EC 5.3.1.5) and xylulokinase (EC 2.7.1.17) are two key enzymes in xylose metabolism (2, 18). To elucidate whether the high xylose utilization is related to a high degree of expression of xylose isomerase in cultured BOH3, gene expression analysis was conducted on strain BOH3 and results were compared to those for the low-xylose-utilizing solventogenic Clostridium sp. strain G117 (producing 5.2 g/liter butanol from 60 g/liter xylose) (9, 36). During the acidogenic phase (0 to 20 h), the expression level of xylose isomerase in strain BOH3 increased up to 165-fold, which is 9 times higher than that of strain G117 (Fig. 5). The enhanced transcription of the xylose isomerase gene in strain BOH3 supports its higher xylose utilization capability compared to those of other solventogenic strains, such as strain G117.

FIG 5.

Comparison of relative xylose isomerase transcription levels between Clostridium sp. strain BOH3 and Clostridium sp. strain G117 in a medium containing 60 g/liter xylose as a carbon source. Xylose isomerase mRNA-cDNA copy numbers were first normalized against the mRNA-cDNA copy numbers of housekeeping genes (fabZ for strain BOH3 and pepT for strain G117) at each time point. The fold transcript increment then is calculated by normalized xylose isomerase copy numbers at each time point divided by normalized xylose isomerase copy numbers at the 8-h time point. Values are averages from duplicate determinations.

In previous studies, xylose isomerase and xylulose kinase were induced only by the presence of xylose, and their expression was inhibited by glucose, a phenomenon referred to as carbon catabolite repression (13). To further elucidate the underlying reason for the BOH3 strain's efficient utilization of xylose and simultaneous utilization of glucose and xylose, activities of xylose isomerase and xylulose kinase were measured in the cell extract obtained at the cultures' exponential growth phase (36 h) with various amounts of glucose and xylose (i.e., glucose alone, glucose and xylose mixtures at different ratios, and xylose alone) (Table 2). As expected, almost no xylose isomerase and xylulose kinase activities were observed in the samples fed solely with glucose. For cultured BOH3, the increments of enzymatic activities of xylose isomerase (0.25 to 0.66 U/mg protein) and xylulose kinase (0.46 to 0.84 U/mg protein) are consistent with the increased amount of xylose in the glucose-xylose mixtures (from 2:1 to 1:2), and the highest activities of xylose isomerase (0.97 U/mg protein) and xylulose kinase (1.16 U/mg protein) were reached when xylose was the sole carbon source (Table 2). For cultured G117, the highest activities of xylose isomerase (0.42 U/mg protein) and xylulose kinase (0.76 U/mg protein) were reached when xylose was the sole carbon source. However, the activities of xylose isomerase and xylulose kinase were severely inhibited when glucose was present in the medium. For example, the xylose isomerase and xylulose kinase activities for strain BOH3 are 0.43 and 0.63 U/mg protein in a glucose and xylose mixture of 30 g/liter:30 g/liter, while the activities for strain G117 are only 0.09 and 0.12 U/mg protein, respectively. When fed with 60 g/liter xylose only, the xylose isomerase and xylulose kinase activities for strain BOH3 increased to 0.97 and 1.16 U/mg protein, whereas these were only 0.42 and 0.76 U/mg protein for strain G117. The increments of xylose isomerase and xylulose kinase activities for strain BOH3 are proportional to the xylose ratio, while these two enzymatic activities for strain G117 are strongly inhibited by glucose. These findings suggest that expression of xylose isomerase and xylulose kinase genes in strain BOH3 are not repressed in the presence of glucose.

TABLE 2.

Specific enzymatic activities (U/mg protein) of xylose isomerase and xylulokinase associated with various glucose/xylose ratios in Clostridium sp. strain BOH3 and Clostridium sp. strain G117

Substrate	Activity ofa:
	Xylose isomerase		Xylulokinase
	Strain BOH3	Strain G117	Strain BOH3	Strain G117
Glucose (60 g/liter)	NDb	ND	ND	ND
Glucose:xylose (40 g/liter:20 g/liter)	0.25 ± 0.12	0.06 ± 0.01	0.46 ± 0.05	0.09 ± 0.02
Glucose:xylose (30 g/liter:30 g/liter)	0.43 ± 0.01	0.09 ± 0.02	0.63 ± 0.11	0.12 ± 0.04
Glucose:xylose (20 g/liter:40 g/liter)	0.66 ± 0.11	0.10 ± 0.05	0.84 ± 0.06	0.16 ± 0.06
Xylose (60 g/liter)	0.97 ± 0.12	0.42 ± 0.07	1.16 ± 0.15	0.76 ± 0.01

^aThe data are calculated from triplicates and are represented as averages ± standard deviations. The samples were taken at a time point of 36 h.

^bND, not detected (the detection limit is <0.01).

DISCUSSION

In this study, the newly reported Clostridium sp. strain BOH3 shows efficient xylose utilization and cofermentation of glucose and xylose simultaneously, which are supported by (i) generating a larger amount of butanol (14.9 versus 14.5 g/liter) and riboflavin (110.5 mg/liter versus 76.8 mg/liter) from xylose (60 g/liter) than glucose (60 g/liter); (ii) full utilization of glucose and xylose in glucose-xylose mixtures and hydrolysates from horticultural wastes; and (iii) higher expression of the xylose isomerase gene and higher enzymatic activities of xylose isomerase and xylulose kinase, responsible for xylose utilization. Moreover, compared to previously reported wild and mutant strains in batch fermentations fed with 60 g/liter xylose, strain BOH3 showed the highest xylose utilization (100%) and butanol production (14.9 g/liter) (Table 1). Therefore, among all of the reported solventogenic microorganisms, simultaneous utilization of glucose and xylose enables Clostridium sp. strain BOH3 to produce the largest amount of butanol (12.4 g/liter) from glucose and xylose mixtures under similar growth conditions (14, 37, 38).

During the fermentation process, strain BOH3 coproduces riboflavin, which adds to the economic value of the process. Usually, a major concern for a coproduction process is whether the second product affects the yield of the major product. For strain BOH3, high butanol production (14.9 g/liter) still occurred, accompanying 110.5 mg/liter riboflavin generation, which is close to its saturation amount (120 mg/liter) in the medium (26, 32). Therefore, results in this study support the rationale of coproduction of another value-added product in the ABE fermentation process so as to make it more economically feasible. However, levels of butanol and riboflavin produced from the hydrolysates of horticultural wastes are still lower than those from a pure monosaccharide mixture. It is known that furfural and hydroxymethylfurfural present in lignocellulosic hydrolysates can be converted to their alcohol or acid derivatives by microorganisms through NAD(P)H/NAD(P)⁺-dependent redox reactions (1, 40). These processes may break the cofactor balance in sugar metabolism of Clostridium to affect butanol formation (1). In addition, other components (e.g., vanillin) in the hydrolysate may also affect the sugar transport system (1, 40).

For solventogenic Clostridium, xylose is usually transported into cytoplasm initially by a xylose transporter and then is further converted into xylulose-5-phosphate by xylose isomerase and xylulose kinase (18, 37, 38). Strengthening this xylose transport system in C. acetobutylicum ATCC 824 and C. beijerinckii NCIMB 8052 has been proven to partially improve xylose uptake; however, they still show lower xylose utilization than glucose utilization, and carbon catabolite repression still exists in C. acetobutylicum ATCC 824 (37, 38). For example, xylose was still left at 13 g/liter after glucose was fully utilized in a 40 g/liter glucose-20 g/liter xylose mixture when using gene-modified C. acetobutylicum ATCC 824 (overexpression of xylT and inactivation of phosphoenolpyruvate [PES]-dependent phosphotransferase system [PTS]) (37). By further overexpression of xylose isomerase and xylulose kinase, C. acetobutylicum ATCC 824 consumed nearly all of the xylose (20 g/liter) in the presence of glucose (40 g/liter) and improved butanol production from 7.9 g/liter to 9.1 g/liter, although the bottleneck still existed because of its low butanol tolerance in xylose (37). In contrast, strain BOH3 shows a higher transcription level of xylose isomerase and enzymatic activities of xylose isomerase and xylulose kinase when using xylose alone than those of the low-xylose-utilizing strains, such as G117 (Table 2). Most importantly, the activities of xylose isomerase and xylulose kinase in strain BOH3 are not inhibited even in the presence of glucose. In contrast, their enzymatic activities in strain G117 were strongly inhibited by glucose (Table 2). As is known, the inhibition of expression of genes encoding enzymes responsible for catabolism of the carbon source over the preferred ones is the main cause of carbon catabolic repression. Moreover, the higher butanol tolerance (15.2 g/liter) of strain BOH3 (6) than for previously reported wild-type microbes (e.g., 9.0 g/liter for C. acetobutylicum ATCC 824 and 7.0 g/liter Clostridium sp. strain G117) could efficiently alleviate the butanol inhibitory effect on the energy-requiring xylose transport system located in the cell membrane (4, 24, 34). Therefore, the high expression of xylose degradation enzymes (xylose isomerase and xylulose kinase) in the presence of glucose contributes to its efficient xylose consumption and relief from carbon catabolite repression.

Conclusions.

Clostridium sp. strain BOH3 shows unique capabilities in simultaneously consuming glucose and xylose to produce butanol at yields of up to 14.9 g/liter with riboflavin as a coproduct. Therefore, strain BOH3 is a promising candidate in transforming lignocellulosic biomass into biofuel due to its efficient use of both xylose and glucose.