Sweet sorghum bagasse as an immobilized carrier for ABE fermentation by using Clostridium acetobutylicum ABE 1201

Abstract

In this study, sweet sorghum bagasse was used as an immobilized carrier for acetone–butanol–ethanol (ABE) fermentation production. Scanning electron microscopy revealed the relationship between Clostridium cells and the sorghum bagasse in terms of adsorption and embedding. The ABE productivity and yield of ABE solvents in batch fermentation were 0.37 g L⁻¹ h⁻¹ and 0.41 g g⁻¹, with 68% and 24% improvement than free cells, respectively. Repeated batch fermentation was carried out under optimized conditions, plus a total of 970 h of continuous fermentation at different dilution rates. A maximum ABE concentration of 16.5 g L⁻¹ was obtained at a dilution rate of 0.08 per h, with optimized yield and productivity. This novel immobilization method using sweet sorghum bagasse offers an attractive prospect for the industrial production of bio-based butanol.

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1. Introduction

Due to the depletion of fossil fuel resources and the increasing price of crude oil, closer attention has been paid during recent decades to alternative sources.¹ Since it offers higher energy density, an improved air–fuel ratio, a lower research and motor octane number,² biobutanol has become recognized as a superior biofuel to ethanol,³ and in particular its convenient production by acetone–butanol–ethanol (ABE) fermentation. However, limited by end-product toxicity, traditional ABE batch fermentation based on free cell culture has suffered from low cell density, low productivity and relative high down-time, resulting in competitive weakness compared with traditional petrochemical-based butanol.^4–6 A further weakness of ABE fermentation is that the strains used gradually lose the ability to produce solvent during repeated inoculated or continuous cultivation.^7,8 It has also been found that concentrated microbial cells are important for high volumetric productivity.⁹

In order to achieve high cell density in ABE fermentation, a technique known as cell immobilization has been widely applied. This technique can help bacteria adhere to the surface of the immobilized carrier by electrostatic adsorption or by embedding bacteria on the interstices of the immobilized carrier surface, which maintains a highly viable cell density in the bioreactor.¹⁰ It is also an ideal way to eliminate downtime of fermentation and keep the strains stable at high productivity.⁹ Previous studies have suggested that a variety of support matrices for immobilization can not only improve ABE output, but also show potential for integrating solvent separation.^11,12 Among these immobilized carriers, a variety of natural lignocellulosic materials, such as wood pulp, corn stalk and sugar cane bagasse, have been examined as natural supports.^1,13,14 However, many of these supports have drawbacks due to their non-uniform structure, which hinder fermentation due to the poor immobilization of the cells.¹⁵ More importantly, it has been difficult to find an ideal immobilization carrier which achieves at the same time both high ABE concentration and high productivity, which at the same time would result in lower purification costs and enhanced process efficiency.

Sweet sorghum, which provides more raw fermentable sugar under marginal conditions than other sugar crops, is considered to be a competitive industrial crop for biorefinery application,^16,17 and has attracted increasing attention recently.¹⁸ According to previous studies, the biological composition and structure of sweet sorghum fibre is quite different from that of other related crops such as corn stalk or sugar cane.^19–21 Compared with other lingo-cellulosic materials, sweet sorghum bagasse can therefore be considered an excellent immobilization carrier on account of its unique performance and superiorities. Our previous study has confirmed that sweet sorghum bagasse is a good immobilization carrier for ethanol production, known as the fungal fermentation system.^15,22 Nevertheless, as far as the authors are aware no studies have previously been performed using sweet sorghum bagasse as a bacteria-immobilized carrier for ABE fermentation, because an immobilized carrier in the bacterial fermentation system has such different characteristics when compared with the use of fungi.

The objective of the present study was to evaluate the applicability and performance of sweet sorghum bagasse as a carrier for immobilization of Clostridia. The size and loading of the carrier for ABE fermentation have been optimized and the performance of repeated batch-fed immobilized fermentation studied. Additionally, the effectiveness of continuous one-stage immobilized ABE fermentation with different dilution rate has been investigated.

2. Experimental

2.1 Materials

Sweet sorghum was cultivated on an experimental plot by the Chinese Academy of Agricultural Sciences at Shunyi District, Beijing. After squeezing out the fermentable juice, the skin and the outside fibre were removed. The bagasse was then soaked in water until the residual sweet juice was washed out, which was designed to eliminate interruption in fermentable sugars. The chopped stalk was then dried, approximately 70 mL moisture being vaporized from each 100 g stalk. The dried bagasse was then sieved to remove both fine and larger particles.

2.2 Strain and culture conditions

In this study the C. acetobutylicum strain ABE 1201 derived from ATCC 824 was used.²³ The culture for ABE fermentation was prepared as described in our previous study, containing 60 g L⁻¹ glucose, phosphate buffer containing 1 g L⁻¹ KH₂PO₄/K₂HPO₄, and 1 mg L⁻¹ p-aminobenzoic acid and 0.01 mg L⁻¹ biotin as vitamins, and in addition to these, 2.2 g L⁻¹ ammonium acetate and minerals for bacteria growth were added (0.2 g L⁻¹ MgSO₄·7H₂O, 10 mg L⁻¹ MnSO₄·H₂O, 10 mg L⁻¹ FeSO₄·7H₂O). After loading the carriers into the medium, ammonium acetate and vitamins were sterilized by separate filtration, and oxygen free N₂ was passed over the surface of the medium for 15 min to maintain an anaerobic environment. After 25 min sterilization at 116 °C, fermentation was started by inoculation of 10% active cells at 37 °C without stirring.

2.3 Batch fermentation

Batch fermentation was carried out at 37 °C in a 1 L glass bioreactor with 600 mL fermentation volume. The bioreactor contained sweet sorghum bagasse of different sizes and ratios of liquid to carrier. The medium was then inoculated with 10% of highly motile cells of C. acetobutylicum ABE 1201. During fermentation, samples were removed at regular intervals to determine the concentrations of acetone, butanol, ethanol and residual sugar. The yield was calculated as the ratio of the total solvent produced to the total glucose utilized.⁴⁰

All the batch operations were carried out in duplicate and the results reported are the mean of two fermentations.

2.4 Repeated batch-fed fermentation

For repeated batch fermentation the method of Yu et al. was employed, using a 1 L glass bioreactor with 600 mL working volume.¹⁵ After 60 h batch fermentation most of the fermentation broth was pumped out and the immobilized carrier with hyper-strains was retained in the fermentor as seed. Fresh medium was then pumped into the fermentor and batch fermentation progressively continued. The immobilized cell system was repeated for 10 feeding cycles over 610 h. During the fermentation process samples were taken at regular intervals to determine the concentrations of acetone, butanol, ethanol and residual sugar.⁴⁰

2.5 Continuous fermentation

The continuous fermentation was performed as in the previous study, with slight modifications.¹ Single-stage continuous immobilized fermentation was used. The bioreactor contained sweet sorghum bagasse (size, 1–2 cm) at a 1 : 20 (w/v) ratio of carrier material to liquid. The fermentation was allowed to proceed in batch mode for 48 h, and the sterilized medium was continuously pumped into the bioreactor at different dilution rates. The working volume of the fermentor was maintained at constant feed rate by removing excess medium from the bioreactor, and fresh medium was continuously fed in using a peristaltic pump (Longer Precision Pump Co., Ltd., Baoding, China). Dilution rates varied from 0.08–0.32 h⁻¹, at a reactor temperature of 37 °C. A steady state was confirmed by stable product values at a specific dilution rate. When the system reached the steady state, solvent productivity was calculated as the total solvents (the sum of A, B and E), or the butanol produced multiplied by the dilution rate. At this point, the dilution rate was defined as the ratio between the feed flow rate and the reactor volume (600 mL).⁴⁰

2.6 Analytical methods

A high performance liquid chromatogram (HPLC) equipped with a refractive index detector (RID; Shimadzu LC-10A, Japan) was used to determine the concentration of residual glucose contained in the fermentation broth. An Aminex HPX-87P carbohydrate analysis column (Bio-Rad Labs, USA) was operated at 65 °C, employing deionized water as the mobile phase at a flow rate of 0.6 mL min⁻¹.

The fermentation products, including acetone, butanol, ethanol, acetic acid and butyric acid, were detected by gas chromatograph (GC, Shimadzu GC-2010, Japan), equipped with a flame ionization detector (FID) and a 2 m glass column packed with Porapack Q 80/100 mesh. Nitrogen was used as the carrier gas. Both injector and detector temperatures were 230 °C, and the column temperature was held at 120 °C for 0.5 min, increasing at 20 °C min⁻¹ to 180 °C, holding for 3 min at 180 °C, increasing at 30 °C min⁻¹ to 230 °C, and holding 10 min at 230 °C. A standard external method was used to determine the respective concentrations of solvents.

2.7 Analysis of the carrier structure

The structure of the carrier was studied using a scanning electron microscope (SEM). Pretreatment of the immobilized carrier was similar to the method used in our previous study.^15,22 The carriers were immersed in 3.5% glutaraldehyde for 6 h, and then dried successively using 50%, 70%, 90%, 95% and 100% ethanol, respectively, followed by overnight retention in a desiccator to remove moisture. The samples were scanned and photographed using a scanning electron microscope (Hitachi Su1510, Japan). The specific surface area was determined standard BET apparatus (V-Sorb 2800S Series, Jinaikang Co. Ltd, Wuhan, China).

3. Results and discussion

3.1 Analysis of immobilization carrier

Ligno-cellulosic materials, although acting as supports in almost all immobilization methods, have limitations in operational stability due to poor cell desorption.¹⁵ The immobilization efficiency and cell absorption on to sweet sorghum bagasse were examined using SEM (Fig. 1). Compared with the clean surface of the non-inoculated alveolate structure shown in Fig. 1A, the sweet sorghum bagasse showed good cell adherence due to its alveolar structure, as in Fig. 1B. Since roughness of the carrier was recognized to be the main factor in the functioning of immobilized systems,²⁴ the surface structure of sweet sorghum bagasse provided an ideally large specific surface area, so that the cells could easily become attached and grow on its porous surface. In this way, the contact surface of the substrate to microorganisms was greatly increased and improved the cell culture density.²⁵ Additionally, the porous surface of sweet sorghum bagasse formed a favourable extracellular microenvironment for promoting cell proliferation and metabolism.²⁶

Fig. 1 Scanning electron microscope images of immobilization carriers: (A) alveolate structure of the carrier before loading C. acetobutylicum; (B) structure within the alveolate, showing a more glabrous surface of the bagasse with a number of pores and wrinkles; (C) C. acetobutylicum cells adhering to the rough surface of sweet sorghum bagasse; cells were firmly immobilized in the recesses of the stalk cells, the wrinkled structure of the bagasse providing a large area for cell adherence, while the alveolate structure could also provide a stable microenvironment for the metabolism of the strains; (D) the specific surface area per unit mass of sugar cane bagasse, corn stalk bagasse and sweet sorghum bagasse. The structure of sweet sorghum bagasse, with its large surface area, was unique in its ability to contribute to the metabolism of the strains.

Since the specific surface area of the bagasse alveolate structure could affect both the adherence of strains to the immobilized carrier and the inner microenvironment for cell metabolism, the specific surface area of different types of sweet stems was further investigated. As illustrated in Fig. 1D, compared with two other bagasses, sweet sorghum bagasse provided an unusually large specific surface area per unit mass (13.78 m² g⁻¹). When used as immobilized carrier in the ABE fermentation process, this large specific surface area would increase the probability of adherence of the strains, contributing to the stability of the microenvironment for cell metabolism.

3.2 Comparison of batch fermentation in the free cell and immobilized cell systems

Immobilized cell technology was found to have a number of advantages over conventional systems, and displayed increased productivity compared to free cells.^27,28 On the other hand, some immobilized systems gave lower productivity. The reason that the physiology of immobilized cells differed from that of free cells was due to a number of factors,²⁹ including nutrient limitations and microenvironments surrounding the cells. These factors have been recognized as key to the change in cell physiology and morphology after immobilization.³⁰ Thus, the parameters of immobilized fermentation might be sharply different from those of free cells.

As shown in Fig. 2, experiments were carried out at the same initial sugar concentration of 60 g L⁻¹ to provide a comparison of batch fermentation in the free cell system and in an immobilized cell system. Almost all the initial sugar in the two systems was utilized, with 0.33 g ABE per g sugar consumed in the free cell system and 0.41 g ABE per g in the immobilized system. In the free cell system, after 88 h of fermentation, a total of 19.21 ± 0.26 g L⁻¹ ABE (2.08 ± 0.16 g L⁻¹ of ethanol, 5.21 ± 0.21 g L⁻¹ of acetone and 11.92 ± 0.22 g L⁻¹ of butanol) was yielded, and the productivity of ABE was ∼0.22 g L⁻¹ h⁻¹. In sharp contrast, the fermentation period was ended after 63 h in the immobilized cells system with an ABE yield of 23.26 ± 0.18 g L⁻¹ (2.27 ± 0.32 g L⁻¹ of ethanol, 6.97 ± 0.21 g L⁻¹ of acetone and 14.02 ± 0.29 g L⁻¹ of butanol), while the productivity of ABE in the immobilized cells system was ∼0.37 g L⁻¹ h⁻¹. The fermentation period in the immobilized cell system was thus almost 28.4% shorter than that in the free cell system, which agrees with previous studies using different types of immobilized carriers.^31–33 With regard to ABE productivity, with a shorter fermentation period and higher ABE concentration, the productivity of ABE solvents in the immobilized cell system was 1.68 times higher than that in the free cell system.

Fig. 2 Comparison of the fermentation kinetics of immobilized cells system and free cells system at an initial sugar concentration of 60 g L⁻¹.

If the volumetric productivity of biobutanol was increased by 50%,³⁴ the cost of biobutanol would become similar to that of synthetic butanol. Woods (1995) has similarly stated that ABE fermentation should be practicable for industrial production if the final solvent concentration could be increased by one-third (i.e. to a level of 22–28 g L⁻¹), and the batch fermentation time could be kept between 40 to 60 h.³⁵ Similarly, when comparing these scenarios it is found that the proposed process, using immobilized ABE fermentation and sweet sorghum bagasse as immobilized carrier, becomes industrially viable.

3.3 Effect of size and solid loading on the immobilized cell system

Most of the immobilized cells are embedded in the interstices of the stalk cells.³⁶ At the same total mass, a larger sorghum bagasse has more intact stalk cells than a smaller one, and as a result more cells can be immobilized in each unit. However, with increasing size of carrier the mass transfer in the interior of the sorghum bagasse will become more difficult due to increasing inner mass transfer resistance, and this will finally influence fermentation productivity.¹⁵ As illustrated in Fig. 3, the effect of immobilized carrier was very obvious. The optimal size of the sorghum bagasse was 1.5 cm × 1.5 cm × 1.5 cm, and under these conditions about 13.88 ± 0.38 g L⁻¹ of butanol and 23.87 ± 0.31 g L⁻¹ of total ABE were yielded after 60 h of fermentation.

Fig. 3 Effect of sorghum bagasse sizes on immobilized cell system.

Solid loading is another key variable in the immobilized cell system.³⁷ In batch fermentation systems solid loading would affect the heterogeneity of the immobilized cell populations and the mixing of the multiphase immobilized cell reactor. Fig. 4 shows the influence of solid loading on the immobilized cell system. The result indicates that the optimal solid loading of immobilized ABE fermentation was 1 : 20 (w/v), and about 14.28 ± 0.35 g L⁻¹ of butanol, 7.39 ± 0.36 g L⁻¹ of acetone and 2.73 ± 0.37 g L⁻¹ of ethanol (total ABE, 24.4 ± 0.28 g L⁻¹) were produced from 60 g L⁻¹ glucose consumed over 60 h. Once the solid loading fluctuated, a correlation between lower productivity and solvents became apparent. This might be attributed to small variations in loading and specific gravity, which have been reported to have a significant influence on air flow rate and mixing time in immobilized systems.³⁸

Fig. 4 Effect of solid loading in the immobilized cell system.

3.4 Repeated batch-fed fermentation in the immobilized cell system

Repeated batch-fed fermentation was further carried out using the optimized conditions to evaluate the long-term stability of the immobilized cell system (Fig. 5). The product profiles in the fermentation broth were found to be similar to those in the previous batch experiment. As expected, repeated batch fermentation with 44–48 g L⁻¹ initial glucose in each batch produced 10–12 g L⁻¹ butanol, 6.5–7.5 g L⁻¹ acetone and 1–1.5 g L⁻¹ ethanol (total ABE, 18–21 g L⁻¹). The overall product yields (g g⁻¹) based on glucose consumed were: butanol 0.23–0.25, acetone 0.15–0.16, ethanol 0.02–0.03 (total ABE, 0.41–0.44), which were similar to batch immobilized fermentation.

Fig. 5 Results of repeated batch fermentation by sweet sorghum bagasse with immobilized cells.

Clearly, repeated batch-fed immobilized fermentation of butanol with sweet sorghum bagasse as carrier gave long-term stability. This might have been caused by the fact that immobilized cells contained a much higher percentage of saturated fatty acids compared with free cells, which led to greater product tolerance in the immobilized cells. Hence, greater survival and cell activity in subsequent cycles compared to free cells could be observed.³⁹ On the other hand another theory for the long-term fermentation activity could be that the immobilized cells might be retaining enzyme activity for a longer time due to their different composition compared to free cells.⁴⁰

3.5 Continuous fermentation at different dilution rates

A continuous system of immobilized cells gives a promising technique that may improve solvent productivity, with the potential for on-line separation.¹¹ However, previous studies of continuous and batch-fed fermentation have found an intrinsic hindrance to high butanol productivity.⁴¹ Low substrate concentrations shifted the system into an acidogenic phase rather than yielding solvents.⁴² The dilution rate of the continuous operation was a possible influencing factor. Alteration of the dilution rate would affect both utilization of glucose and growth of cells in immobilized system,⁴³ resulting in sharp changes in ABE productivity and concentration.^1,14 An opposite dilution rate therefore plays an important role in ABE fermentation.

The result of continuous fermentation at different dilution rates is shown in Fig. 6. In order to protect strains from butanol, known as the end-product inhibition effect, the continuous fermentation system was carried out at a butanol concentration in the fermentation broth below 10 g L⁻¹.⁵ Initially, the medium was fed into the immobilized system at a dilution rate of 0.08 h⁻¹ with 60 g L⁻¹ glucose contained in fresh medium. In this case, the maximized total ABE concentration in the fermentation broth was maintained at ∼16.5 g L⁻¹ (∼9 g L⁻¹ butanol, ∼6 g L⁻¹ acetone and ∼1.5 g L⁻¹ ethanol). The productivity of ABE at the dilution rate of 0.08 h⁻¹ was ∼1.32 g L⁻¹ h⁻¹. As the dilution rate gradually increased, the residual sugar remaining in the fermentation broth also increased. At the same time, the concentration of ABE solvents decreased, which was similar to the tendency reported in previous studies by Survase et al. and Zhang et al. using lignocellulosic immobilized carriers.^14,46 In the present study, when the dilution rate reached 0.32 h⁻¹ there was still ∼40 g L⁻¹ of glucose remaining unfermented. At the same time, the concentration of ABE solvents remained above ∼7 g L⁻¹ (∼4 g L⁻¹ butanol, ∼2 g L⁻¹ acetone and ∼1 g L⁻¹ ethanol), while the highest overall productivity of ABE was obtained (∼2.24 g L⁻¹ h⁻¹) based on the current test.

Fig. 6 Continuous ABE fermentation with different dilution rate in using sweet sorghum bagasse as immobilizated carrier.

3.6 Comparison with other studies

Immobilized cell technology operated in the ABE fermentation process has been widely studied as an effective method to achieve high butanol productivity and system stability, which has promise in online butanol recovery and industrialization of the process. Table 1 summarizes and compares recent studies on immobilized ABE fermentation in using different types of the strains. It is seen that cell immobilization can significantly increase reactor productivity due to increased cell density and the elimination of reactor downtime.⁵ The immobilization technology has provided an in-depth understanding of the different carrier properties of microbial cells. Ligno-cellulosic biomass is seen to be the most cost-effective and sustainable feedstock, and this is more commonly used as a basic immobilization material.^50,51 In the present study sweet sorghum bagasse, the lignocellulosic residue of the ethanol fermentation process, was recovered without chemical modification, for both environmental and economic reasons.¹⁷ Compared to other immobilized methods, with an average ABE yield of 0.3–0.4 g g⁻¹, sweet sorghum bagasse carrier gave more efficient sugar efficient conversion (0.43–0.44 g g⁻¹) in both batch and continuous production.

Table 1 Comparison of batch and continuous reactor performance with different cell immobilization techniques

Scenario	Support material	Strain	ABE production (g L^−1)	ABE yield (g g^−1)	ABE productivity (g L^−1 h^−1)	Operation days	References
Batch	Alkali-treated steam-exploded corn stover	C. acetobutylicum ATCC824	16.95	0.32	0.24	—	25
	Brick	C. acetobutylicum BCRC 10639	∼18	∼0.31	0.17	—	49
	Sweet sorghum bagasse	C. acetobutylicum ABE 1201	24.4	0.44	0.42	—	Present study
Continuous	Fibrous bed	C. beijerinckii ATCC 55025	8.5	0.53	7.6	22	44
	Wood pulp fibers	C. beijerinckii DSM 6423	5.22 (butanol and isopropanol)	0.30	5.22	27	46
	Brick	C. beijerinckii BA101	7.9	0.38	15.80	25	45
	Brick	C. acetobutylicum BCRC 10639	17.25	0.52 (butanol)	0.46	13	43
	Corn stalk	C. beijerinckii ATCC 55025	5.1	0.32	5.06	20	14
	Polyvinyl alcohol	C. beijerinckii NCIMB 8052	22.1	0.44 (butanol)	0.40	6.5	47
	Wood pulp	C. acetobutylicum DSM 792	12	0.27	4.86	—	48
	Sweet sorghum bagasse	C. acetobutylicum ABE 1201	16.5	0.43	1.32	41	Present study

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In addition, conventional ABE fermentation has the drawback of low butanol productivity, yield and titre, which has led to biobutanol falling behind petrochemical butanol in economic competitiveness.^2,52 Integrating the product recovery process and fermentation is a method of solving the problem.⁵³ In response to the need for an in situ separation processes, the immobilized fermentation process simultaneously gives higher ABE production and productivity, which maximizes the efficacy of process and minimizes the cost of purification. In the present study, the continuous fermentation process at a dilution rate of 0.08 h⁻¹ offered advantages both in ABE production (16.5 g L⁻¹) and productivity (1.32 g L⁻¹ h⁻¹). Other processes, however, showed disadvantages, resulting in higher product recovery costs (due to low ABE concentrations) or lower productive efficiency (due to low ABE productivity).

The immobilized process using sweet sorghum bagasse carrier gave the longest continuous operation time (970 h) in ABE fermentation, showing good stability and economic feasibility. By comparison, in the free cell continuous fermentation process, solvent production started to reduce after only a relatively short time (140 h), due to the poor pH tolerance of the strains.⁵⁴ Thus, it was clear that ABE fermentation using sweet sorghum bagasse as immobilization carrier offered an efficient and stable method for the production of biobutanol.

4. Conclusions

This study has led to a novel immobilization method for Clostridium to sorghum bagasse for butanol production. The immobilized system gave improved ABE production and higher productivity. It is worth noting that the immobilized method could achieve a relatively high ABE yield of 0.43–0.44 g g⁻¹ glucose. Ten batches of repeated batch-fed fermentation were produced continuously, with high stability of the system. In continuous mode, a total of 970 h of long-term fermentation at different dilution rates showed advantages in both ABE production and productivity when using sweet sorghum bagasse as immobilized carrier. The immobilized fermentation process maintained stable productivity and high butanol yield over an extended period, making the process attractive for the industrial production of biobased butanol.

Acknowledgements

The authors wish to express thanks to the National Basic Research Program of China (Grant nos 2013CB733600 and 2012CB725200), the National High-Tech R&D Program of China (Grant nos 2012AA021404 and 2011AA02A205), the National Nature Science Foundation of China (Grant nos 21076017 and 21276017), and the National Key Scientific Instruments and Equipment Development Special Fund (Grant no. 2012YQ0401400302).

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Footnote

† Equal contributors to the paper.

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