DOI:
10.1039/C5RA24923F
(Paper)
RSC Adv., 2016,
6, 33695-33704
Extracellular polymer substances and the heterogeneity of Clostridium acetobutylicum biofilm induced tolerance to acetic acid and butanol
Received
24th November 2015
, Accepted 17th March 2016
First published on 21st March 2016
Abstract
The mechanisms associated with how cells in biofilms exhibit enhanced tolerance to adverse environmental stress have attracted much recent attention. In this study, we investigated the tolerance mechanisms through observation of biofilm morphology combined with detection of fermentation activity, and discovered an improved way to culture biofilms for application in acetone–butanol–ethanol (ABE) fermentation. We found that a mature biofilm exhibited enhanced tolerance to acetic acid and butanol during ABE fermentation. A mature biofilm consists of a complex, heterogeneity three-dimensional structure, with a coated extracellular polymer substance (EPS). Therefore, when exposed to a harsh environment, cells in different regions of the biofilm displayed different levels of performance, resulting in cells with higher tolerance levels capable of survival, continued growth. The EPS acted as a barrier, limiting the transfer of harmful substances, and diluting their concentration in order to protect biofilm cells. During repeated-batch fermentations, the continuous fermentation formed biofilms, and the butanol concentration, productivity, and yield were 22.08%, 26.37%, and 61.08% higher, respectively, relative to suspended fermentation.
1. Introduction
Cell immobilization onto surfaces and formation of biofilms are important for processes like microbial fuel cells,1 wastewater treatment,2 continuous fermentation,3 bacterial infection,4 etc. Biofilms used in continuous fermentation offers advantages, such as increased fermentation productivity, feasibility of continuous processing, cell stability, and lower costs associated with recovery, recycling, and downstream processing, making the technology widely used in industrial fermentation.5,6 Cells can be immobilized by adsorption, entrapment, and covalent-bond formation.7 Biofilms immobilize cells by adsorption, with cells irreversibly or reversibly attached to solid surfaces, and embedded in a self-produced extracellular polymeric substances (EPS) matrix to form the biofilm.8 Biofilm reactors exhibit high biomass density, stability, and potential for long-term fermentation, with reduced substrate concentrations and continuous product removal.9 Moreover, cells in biofilms exhibit enhanced tolerance to adverse environmental stress conditions, which has increased recent interest in biofilm use for biotechnological fermentation.10,11
Biofilm has a complex three-dimensional structure that creates an internal protective environment for bacterial cells.12 The EPS is capable of slowing diffusion of harmful substances, helping the biofilm architecture maintain inherent heterogeneity and complexity, and generating large variations in biofilms for different conditional responses.13–15 However, the exact mechanism associated with this process is poorly understood.16 The tolerance of bacterial biofilms to physical stress, chemical agents, and toxic solvents depends upon several intrinsic parameters and their interactions.17,18 Understanding the mechanisms associated with this tolerance will enable application of immobilized technology in fermentation.19
Butanol has the advantages of high energy content, low volatility, and reduced levels of hygroscopicity and corrosiveness to existing infrastructure. Butanol shares similar characteristics with gasoline, and can be used directly in any gasoline engine without modification and/or substitution.20 Thus, biotechnological production of butanol has become a research hotspot since the early 20th century. The high price of feedstocks, low butanol yield and titers, and low bacterial tolerance to solvent inhibit economically competitive butanol production as compared to petro-chemical methods.21,22
In order to solve these problems, researchers have investigated methods to allow low-cost solutions, including using alternative feedstocks (such as lignocellulosic residues) or designing targeting genes, enzymes, or pathways, to improve butanol yield and/or increase the solvent resistance of bacteria.23 Other studies have focused on in situ butanol removal technologies, such as gas stripping, pervaporation, or adsorption, to alleviate butanol toxicity in cells and improve the productivity of acetone–butanol–ethanol (ABE) fermentation systems.24 While these efforts have resulted in limited success, it remains difficult to meet the demands of industrial production. Cell immobilization is a more advantageous method for continuous operation of large-scale butanol production as a biofuel.25 Clostridium acetobutylicum is an efficient butanol-producing bacterium capable of forming biofilms with increased tolerance to toxicity; however, the mechanism associated with this increased tolerance remains unclear. So many researchers have focused on exploring the tolerance mechanisms associated with biofilm formation in the fermentation process in order to discover suitable methods for optimally culturing biofilms for butanol production.19
During ABE fermentation, the lipophilic solvent butanol disrupts the phospholipid components of the cell membrane, causing increased membrane fluidity and inhibition of both cell growth and glucose uptake by lowering ATPase activity.11,26,27 If the acid concentration is too high, acid crash occurs and solvent production stops.27 In this study, we investigated cellular tolerance following the addition of butanol and acetic acid products at different periods in the fermentation process. We discussed the mechanism by comparing cell morphology and fermentation activity and determined the optimal tolerance growth phase of cells and biofilm to achieve appropriate biofilm development. The methods described here enabled continuous fermentation, even in harsh environments.
2. Materials and methods
2.1. Bacterial culture and growth conditions
Here, we used C. acetobutylicum CGMCC 5234 preserved in our laboratory. The strain was stored in 30% (v/v) glycerol at −80 °C, and was cultured in modified P2 medium containing 10 g L−1 glucose as the sole carbohydrate source for seeding at 37 °C in an anaerobic chamber. The fermentation medium contained 60 g L−1 glucose, 0.5 g L−1 K2HPO4, 0.5 g L−1 KH2PO4, 2.2 g L−1 CH3COONH4, 0.2 g L−1 MgSO4·7H2O, 0.01 g L−1 MnSO4·H2O, 0.01 g L−1 NaCl, 0.01 g L−1 FeSO4·7H2O, 1 mg L−1 p-aminobenzoic acid, 1 mg L−1 thiamine, and 0.01 mg L−1 biotin, and was incubated at 37 °C with 10% inoculum (v/v) in anaerobic atmosphere.
2.2. Cell immobilization and different fermentation patterns
Screw-capped bottles (250 mL) were used as reactors for anaerobic fermentation. The C. acetobutylicum CGMCC 5234 cells were immobilized by adsorption onto cotton towels (6 cm × 6 cm; specific surface area: >40 m2 m−3; thickness: 5 mm), which were washed twice with deionized water, and dried at 60 °C. 90 mL medium, 10 mL seed culture and a piece of cotton towel were in the bottle and incubated in an anaerobic chamber at 37 °C for simultaneous cell immobilization by adsorption onto cotton towels and fermentation. The suspended-cell fermentation process was the same as the immobilized-cell fermentation process without the cotton towels. Repeated-batch fermentation was in a 2 L fermentation tank. The medium circulation rate was maintained at 35 mL min−1 via a peristaltic pump during fermentation. For repeated-batch immobilized fermentation, using cotton towels fixed in iron as adsorption material for biofilm formation, preventing the necessity for repeated inoculations. The old broth was replaced by new fresh broth when the sugar concentration approached depletion. For repeated-batch suspended fermentation, using a hollow-fiber membrane to retain cells that were then returned to the fermentation tank with the fresh broth.
2.3. Analytical methods
The cell density was analyzed by measuring the OD600 with a spectrophotometer (Model 7200; Unico Instrument Co., Ltd., Shanghai, China). Cells in biofilm were estimated by cell counting using scanning electron microscopy (SEM) at ∼0.65–0.85 CFU μm−2, with a layer of cells immobilized on a cotton towel counted at ∼4.32–5.76 × 108 CFU. Using the hemocytometer method of counting suspended cells, we inferred that 1 U at OD600 was equivalent to ∼1.17–1.79 × 108 CFU. Therefore, a layer of cells was capable of increasing the OD600 by ∼2.42–4.94.
Acetone, ethanol, butanol, and acetic acid were determined by gas chromatography (model 7890A; Agilent Technologies, Santa Clara, CA, USA) equipped with a flame ionization detector, an autosampler (model 7693; Agilent Technologies), and a glass column (HP-INNOWax, 60 m × 0.25 mm × 0.5 μm; Agilent Technologies). The oven temperature was programmed from 70 °C to 190 °C at a gradient of 20 °C min−1, with an initial holding time of 0.5 min and a post-holding time of 4 min. The injector and detector temperatures were maintained at 180 °C and 220 °C, respectively. Samples were first passed through a 0.22 μm syringe filter with an injection volume of 1 μL. Nitrogen was used as a carrier gas at a flow rate of 30 mL min−1.
Glucose was measured at OD540 with a spectrophotometer, and 0.5 mL of the sample and 0.5 mL DNS (mainly containing 3,5-dinitrosalicylic acid, phenol, seignette salt) were mixed together, boiled for 5 min, then cooled in ice water immediately. Deionized water (8 mL) was then added for detection.
2.4. Characterization
2.4.1. SEM. To image the suspended cells and biofilm by SEM, suspended cells and immobilized cells on the cotton towel were fixed in 2.5% glutaraldehyde in 0.2 M phosphate-buffered saline [PBS; pH (7.3–7.4)] for over 4 h and washed twice with deionized water. Cells were fixed again using 1% osmium tetroxide in 0.1 M PBS (pH 7.0–7.5) for 1–2 h and washed twice by deionized water, followed by dehydration in a graded ethanol series. After treatment, the suspended cells were dropped onto the cover glass for natural drying. The samples were sputter coated with gold–palladium for 2 min in a Polaron E5100 Series II sputter coater (Polaron Equipment Ltd., Watford, Hertfordshire, UK), and specimens were viewed by SEM operating at 10 kV.
2.4.2. Confocal laser-scanning microscopy (CLSM). In order to image suspended cells and biofilm by CLSM and to minimize change of morphology during preparation, biofilms were treated with 2.5% glutaraldehyde for 30 min prior to staining. Samples were imaged with a LSM 510 microscope (Carl Zeiss Inc., Thornwood, NY, USA), and images were analyzed using Zeiss LSM image examiner software (Carl Zeiss Inc.). STYO 9/PI stored frozen at ≤−20 °C [3.34 mM SYTO 9 dye (component A) in 300 μL dimethyl sulfoxide (DMSO) solution and 20 mM propidium iodide (component B) in DMSO] was used to label live and dead cells. Samples were suspended in 1 mL deionized water in tubes, and 1 μL of component A and 1 μL of component B were added to each sample tube, following each tube was vortexed, incubated at room temperature or 37 °C, protected from light for 15–30 min, and visualized using an excitation wavelength of 485/535 nm and an emission wavelength of 498/617 nm.Acridine orange was used to visualize biofilm heterogeneity. Acridine orange power (0.1 g) was dissolved in 100 mL deionized water and diluted 10 times using PBS (pH 7.0). Samples were stained for 5 min at room temperature in the dark and visualized at an excitation/emission wavelength of 492/640 nm.
2.4.3. Transmission electron microscopy (TEM). TEM was used for ultrastructural analyses. Samples were fixed as previously mentioned, then prepared in copper grids and stained with 1% aqueous uranyl acetate for 90 s. Following several washes in deionized water, samples were dehydrated by infrared lamp and visualized by a JEOL transmission electron microscope (JEOL 1200 EX USA, Inc., Peabody, MA, USA).
3. Results and discussion
3.1. Bacteria and biofilm growth processes
During fermentation, bacterial densities increase over time, with differences observable between suspended and immobilized cells. As fermentation progressed, the suspended bacteria continued to divide and proliferate, achieving high cell density (Fig. 1a–d). For biofilm characterization (Fig. 1e–h), cells first formed an initial attachment to the surface, then aggregated into microcolonies, followed by microcolony growth culminating in formation of mature biofilm.5,28 We observed that the immobilized cells were shorter and coarser relative to the planktonic cells, which may have been more active (Fig. 2a and b). We also observed substance accumulation around the cells that may have been extracellular matrix secreted by the cells during biofilm formation (Fig. 2c and d). Following alkali treatment, cells were lysed and left many pores, with the resulting mature biofilm consisting of an extensive fibrous network (Fig. 2e and f). These results indicated that cells immobilized on the cotton towels were encased by EPS, forming a three-dimensional network-like architecture containing proteins, polysaccharides, nucleic acids, and lipids, and offering a wide range of advantages to biofilm propagation.29,30
 |
| Fig. 1 SEM images of Clostridium acetobutylicum CGMCC 5234 cultured at different stages. (a–d) Panels show cells in suspended fermented liquid (upper) and (e–h) cells immobilized on cotton towels (lower). | |
 |
| Fig. 2 (a and b) Field emission scanning electron-microscope images of cells in suspended fermentation and biofilm, respectively. (c and d) TEM images of C. acetobutylicum CGMCC 5234 in biofilm. (e and f) SEM images of the biofilm after treatment with 0.1 M NaOH. | |
Fig. 3 shows the density of bacteria and the degree of turbidity of the fermented liquid from the immobilized and suspended fermentations. In the immobilized fermentation, the density of the suspended cells was less relative to that observed from the suspended fermentation (Fig. 3a). However, the number of immobilized cells derived from the SEM images was much larger. Based on macroscopic observation, we observed mucoid substances adhered to the cotton towel (Fig. 3b and d). Fig. 3c shows the turbidity of the fermented liquid, where the liquid from the immobilized fermentation appeared more transparent relative to that of from the suspended fermentation. These results indicated that during immobilized fermentation, more bacteria were capable of adhering to materials for biofilm formation, reaching a high level of bacterial density that was beneficial to the fermentation rate.31
 |
| Fig. 3 (a) The density of cells in suspended fermented liquid (black square), suspended bacteria in the immobilized fermented liquid (red dot), and a biofilm of cells immobilized on a cotton towel in the immobilized fermented liquid (blue triangle). (b) A cotton towel used as an immobilized carrier in the fermentation broth after 36 h (left) and in water (right). (c) The turbidity of the fermented liquid from the suspended (1) and immobilized (2) fermentations and water (3). (d) Magnification of the white square (b). | |
3.2. Effects of acetic acid supplementation
ABE fermentation can be divided into two distinct steps: acidogenesis and solventogenesis. In the first step, the bacteria convert carbon sources into organic acids (acetic acid and butyric acid), decreasing the pH of the fermented liquid and acting as a stress on the bacteria. However, as cells grow, the bacteria exhibit a metabolic switch, where acidogenesis is switched “off”, and initiate solventogenesis, where the acids are converted into organic solvents, such as butanol, acetone, and ethanol.32
The initial concentration of acetic acid was much higher than that of butyric acid.33 Therefore, here, we added acetic acid as a representative of the intermediate to the liquid at different fermentation time, then evaluated the effects on the fermentation activities involving glucose consumption, butanol production, bacterial growth, and biofilm formation. We added acetic acid at fermentation time of 12 h, 18 h, 24 h, and 36 h. In the suspended fermentation, these time represented the end of the delay phase, the middle of the logarithmic phase, the end of the logarithmic phase, and the stationary phase of bacterial growth, respectively. In the immobilized fermentation, these time corresponded to the initial adhesion phase, the accumulation phase, the growth phase, and the mature phase of biofilm formation.
Fig. 4 shows the kinetics of glucose consumption and butanol production in the suspended and immobilized fermentations following addition of acetic acid at different fermentation time. The concentration of acetic acid changed during fermentation due to the synthesis and consumption of acetic and butyric acid during the process. During the fermentation process, the pH was ∼4.5–5.0.33 In order to maintain the same acetic acid concentration under different conditions, we added acetic acid until the pH reached 4.0. Based on the fermentation results, we observed that this treatment resulted in decreased glucose-consumption and butanol-production rates as compared to the control when we added acetic acid at different fermentation time. In both the suspended and immobilized fermentations, adding acetic acid after 12 h caused bacteria to cease consuming glucose and producing butanol. When acetic acid was added at 18 h and 24 h in the suspended fermentation, the rates of glucose consumption and butanol production were much slower relative to the control; however, the rates of immobilized fermentation were only slightly slower relative to the control. When acetic acid was added at 36 h in suspended fermentation, the rates were slightly slower, and the immobilized fermentation performance was almost the same as that observed in the control.
 |
| Fig. 4 (a) The kinetics of glucose consumption (solid line and closed symbol) and butanol production (dashed line and closed symbol) during suspended fermentation and following addition of acetic acid at different fermentation time. (b) The kinetics of glucose consumption (solid line and closed symbol) and butanol production (dashed line and closed symbol) during immobilized fermentation and following addition of acetic acid at different fermentation time. The black, red, blue, magenta, and green lines represent the control and addition of acetic acid at 12 h, 18 h, 24 h, and 36 h time points, respectively. | |
Fig. 5 shows the morphology of suspended cells and biofilm cultured 12 h after the addition of acetic acid. When acetic acid was added to the suspended fermentation at 12 h (Fig. 5a), there were few bacterial cells in the broth. When acetic acid was added at 18 h and 24 h (Fig. 5b and c), we observed the accumulation of a substance around the cells that may have been produced by cell lysis, as well as circular-shaped cells appearing to be spores. Following acetic acid addition at 36 h (Fig. 5d), the cells maintained a normal rod shape. In the immobilized fermentation, adding acetic acid at 12 h (Fig. 5e) resulted in almost no bacteria adhering to the cotton towel. Following acetic acid addition at 18 h, we observed some cells adhesion to the surface, however, no accumulation or growth occurred over the next 6 h (Fig. 5f) relative to the control (Fig. 1g). Following addition of acetic acid at 24 h (Fig. 5g), many cells accumulated and formed the biofilm; however, the biofilm did not reach maturity after 12 h as compared to the control (Fig. 1h). Following acetic acid addition at 36 h (Fig. 5h), we observed formation of mature biofilm that maintained a complete 3D structure after 12 h.
 |
| Fig. 5 SEM images of C. acetobutylicum-suspended cells and biofilms cultured 12 h after the addition of acetic acid. Panels show cells in the suspended fermentation (upper) and biofilm on a cotton towel in the immobilized fermentation (lower). (a and e) The addition of acetic acid at the 12 h fermentation time. (b and f) Acetic acid addition at 18 h. (c and g) Acetic acid addition at 24 h. (d and h) Acetic acid addition at 36 h. | |
We inferred from these data that bacteria in different growth phases exhibited different degrees of tolerance to acetic acid, with the stationary-phase cells showing stronger tolerance and maintaining cell morphology and fermentation activity. In the acidic environment, some cells were dead, but more bacteria were capable of activating the cascade of genes enabling spore formation for survival, resulting in dormant, metabolically inactive bacteria with high resistance,33,34 but low fermentation activity. Compared to suspended cells, the immobilized biofilm showed stronger tolerance, even while in the accumulation phase. Given the presence of cells in different growth phases in the biofilm, the cells exhibiting stronger resistance were able to survive and divide.10 The mature biofilm was minimally affected by the addition of acetic acid. Therefore, we suggested that the mature biofilm with EPS and stable 3D structure was capable of protecting inner cells.12
3.3. Effects of butanol supplementation
One of the most critical problems in ABE fermentation is butanol toxicity. About 4.0–4.8 g L−1 butanol inhibits cell growth, while >13 g L−1 butanol will completely stops cell growth.35 Here, we studied the tolerance of bacteria to butanol in order to determine better fermentation conditions. Given that ABE fermentation produces butanol, in order to reach inhibition, we added ∼3 g L−1 butanol to the broth at fermentation time of 12 h, 18 h, 24 h, and 36 h in order to study the effects of additional butanol on fermentation activity, the morphology of suspended cells, and biofilm formation.
Fig. 6 shows the kinetics of glucose consumption and butanol production following addition of butanol to the suspended and immobilized fermentations at different fermentation time. We observed that adding ∼3 g L−1 butanol at different fermentation time inhibited glucose consumption and butanol production to a certain degree in the suspended and immobilized fermentations.
 |
| Fig. 6 (a) The kinetics of glucose consumption (solid line) and butanol production (dashed line) in the suspended fermentation following addition of butanol at different fermentation time. (b) The kinetics of glucose consumption (solid line) and butanol production (dashed line) in the immobilized fermentation following addition of butanol at different fermentation time. | |
The slope represents the rate. In the suspended fermentation (Fig. 6a), following addition of butanol to the broth after 12 h indicated a final butanol concentration of <4 g L−1, with the butanol production rate slightly lower than that observed in the control. Butanol addition at 18 h and 24 h resulted in final butanol concentrations of 4.49 g L−1 and 4.81 g L−1, respectively, with rates of butanol production and glucose consumption much lower relative to the control. Adding butanol at 36 h resulted in a final butanol concentration of 8.64 g L−1, with rates slightly lower relative to the control. These results suggested that when the butanol concentration reached 4 g L−1, the activity of cells prior to stationary phase were inhibited, while cells in the stationary phase showed stronger tolerance, even at butanol concentrations >8 g L−1.
In the immobilized fermentation (Fig. 6b), adding butanol at 12 h, as the butanol concentration was low, which caused minimal inhibition over the next 6 h, with rapid recovery. When butanol concentration reached ∼12 g L−1, the fermentation ceased butanol production. Adding butanol at 18 h and 24 h resulted in final butanol concentrations of 4.16 g L−1 and 8.59 g L−1, respectively, along with lower glucose consumption and butanol production rates, which decreased linearly as butanol concentration increased. When butanol was added at 36 h, the final butanol concentration reached 12.34 g L−1, and the rate of glucose consumption was similar to that observed in the control, although butanol production almost ceased. These results indicated that in the immobilized fermentation, ∼12.5 g L−1 butanol may have been the limit value for butanol production in the ABE fermentation, and the mature biofilm showed higher tolerance, with less impact by ∼12 g L−1 butanol. Prior to the mature phase, only when the butanol concentration reached 8 g L−1, which would result in obvious inhibition of glucose consumption and butanol production.
As shown in Fig. 7, the morphology of suspended cells and biofilm cultured 12 h after the addition of butanol. In the suspended fermentation, following the addition of butanol and when the butanol concentration was still low, bacterial concentrations were slightly lower than those observed in the control (Fig. 7a, as compared to Fig. 1c). However, once the concentration reached ∼4 g L−1, many cells in the phase prior to the stationary phase appeared dead and lysis (the number of cells were lower in Fig. 7b and c, as compared to Fig. 1c and d). Cells in the stationary phase in the presence of higher butanol concentrations maintained stable cell concentrations and morphology (Fig. 7d). In the immobilized fermentation, following butanol addition and while the butanol concentration was still low, biofilm formation was slightly inhibited, and it retained the capability to continue accumulating (Fig. 7e, as compared to Fig. 1g) and growing (Fig. 7f, as compared to Fig. 1g) relative to the control. However, when the butanol concentration reached 8 g L−1, biofilm formation prior to the mature-growth phase was severely inhibited (Fig. 7g, as compared to Fig. 1h), although the mature biofilm was capable of maintaining an intact structure even the butanol concentration was over 12 g L−1 (Fig. 7h).
 |
| Fig. 7 SEM images of C. acetobutylicum-suspended cells and biofilms cultured 12 h after adding butanol. Panels show cells in the suspended fermentation (upper) and biofilm on a cotton towel in the immobilized fermentation (lower). (a and e) The addition of butanol after 12 h fermentation. (b and f) The addition of butanol after 18 h. (c and g) The addition of butanol after 24 h. (d and h) The addition of butanol after 36 h. | |
Observing the combination of fermentation activity and morphology, we suggested that in the presence of butanol, cells in a biofilm would be able to tolerate a higher concentration of butanol than would bacteria in suspended fermentation. Many suspended cells appeared to die before the stationary phase and caused decreased rates of glucose consumption and butanol production, while cells in biofilm continued to grow, so the fermentation activity was slightly inhibited. The mature biofilm was capable of maintaining fermentation activity in an environment consisting of high butanol concentrations.
3.4. Sporulation alleviates environmental pressure
Spores form when the vegetative organism is subjected to stress, enabling bacteria to survive in a dormant state with low metabolism.33 Fig. 8 shows the changes in cell and biofilm morphology under harsh environmental conditions. After adding acetic acid and butanol during fermentation, many bacteria in the suspended fermentation underwent sporulation, especially in the acidic environment (Fig. 8a and b). However, in the immobilized fermentation, cells aggregated to form a biofilm and exhibited enhanced tolerance, indicating that the spore content was low (Fig. 8c and d).
 |
| Fig. 8 SEM images of C. acetobutylicum morphological changes after being cultured for 12 h under adverse conditions. Panels show cells in the suspended fermentation (upper) and biofilm on cotton towels in the immobilized fermentation (lower). (a and c) Addition of acetic acid until the pH = 4.0. (b and d) Addition of ∼10 g L−1 butanol. | |
3.5. Heterogeneity and EPS act as cell protection mechanisms
From the previous experimental results, we observed that bacteria in different phases displayed different degrees of tolerance to acetic acid and butanol. During biofilm formation, cells in EPS ingested nutrients and produced waste and signaling factors and underwent cell division, growth, and decay. Therefore, different regions of the biofilm displayed different physiological characteristics, indicating heterogeneity.36 Fig. 9 shows the heterogeneous biofilm by CLSM. Different colors reflected different RNA content, which indicated cell-growth rates. We observed that cells in the suspended fermentation displayed a similar yellow hue following acridine orange staining (Fig. 9b), while some areas of the biofilm were red in color, corresponding to high relative RNA content and rapid growth, other areas of the biofilm were yellow, reflecting low relative RNA content and a slower growth rate (Fig. 9a). Under harsh conditions, cells in different phases displayed different sensitivity levels, with those showing higher tolerance capable of survival and continued cell growth.16
 |
| Fig. 9 CLSM images of C. acetobutylicum cells and biofilm, staining with acridine orange allowed visualization of cell activity. (a) Cells in biofilm. (b) Cells in the suspended fermentation. Yellow colour reflect low RNA content and slower growth rates, red colour reflect higher RNA content and faster growth rates. | |
We also found that mature biofilm exhibited the strongest tolerance, and that this property was closely related to its structural integrity and EPS content. Fig. 10 shows cells and biofilm by CLSM following staining with SYTO9/PI after treatment with acetic acid or butanol (green: live cells; red: dead cells). Under harsh conditions in the suspended fermentation, live/dead cells were located throughout the sample (Fig. 10e and f), while in the biofilm, dead cells existed in the external areas of the biofilm, and live cells existed in the bottom and center (Fig. 10c, as compared to Fig. 10a; Fig. 10d, as compared to Fig. 10b). We inferred that the resistance of the biofilm to external treatment may have been due to the limited ability of acetic acid or butanol to penetrate. These results indicated that the mature structure and EPS content protected the inner cells of the biofilm by acting as barriers to chemical transfer.18,37
 |
| Fig. 10 CLSM images of the immobilized biofilm and suspended cells of C. acetobutylicum following SYTO 9/PI staining. (a) Biofilm control without addition of acetic acid or (b) butanol; biofilm following (c) treatment with acetic acid for 8 h and change in pH to 3.0, or (d) treatment with 10 g L−1 butanol for 8 h. (e) Suspended cells after treatment with acetic acid for 8 h and change in pH to 3.0, or (f) treatment with 10 g L−1 butanol for 8 h. | |
3.6. Repeated-batch fermentations with immobilized C. acetobutylicum
Comprehensive experimental results were as follows: we first cultured the suspended cells to the stationary phase, transferred them into a 2 L fermentation tank by using a cotton towel as the immobilization material, and cultured them for ∼36 hours to obtain mature biofilm. During the biofilm formation process, we controlled the pH by buffering during acid production in the fermentation process. The mature biofilms were then utilized for continuous fermentation, resulting in butanol production. And we use a hollow-fiber membrane to hold cells for continuous suspended fermentation. Table 1 shows the average amount of butanol produced from repeated-batch fermentation of immobilized and suspended bacteria. During the immobilized fermentation, butanol concentration, productivity, and yield were 22.08%, 26.37%, and 61.08% higher than suspended fermentation. These results indicated that the immobilized fermentation using mature biofilm was adequate for butanol production.
Table 1 The average productivity, concentration, and yield of butanol from immobilized and suspended fermentation conditions during repeated-batch fermentationa
Process mode |
Butanol concentration (g L−1) |
Productivity (g L−1 h−1) |
Butanol yielda (g g−1) |
Butanol yield was based on the mass ratio of butanol to utilized sugars. |
Immobilized fermentation |
11.386 |
0.201 |
0.948 |
Suspended fermentation |
8.872 |
0.148 |
0.369 |
4. Conclusions
In summary, the present study showed that in acidic environments or those with high butanol concentration, the rate of butanol production and glucose consumption were less affected during immobilized fermentation than during suspended fermentation, and that cells in the biofilm was more capable of maintaining morphology, indicating enhanced tolerance levels relative to those observed in the suspended fermentation. Additionally, by adding butanol or acetic acid at different cell-growth phases, we observed that cells in the stationary phase showed the strongest tolerance. Using CLSM characterization, we revealed that EPS acted as a barrier to exclude harmful substances, and the heterogeneity of biofilm showed different sensitivity to harsh environment, constituting the primary tolerance mechanisms associated with biofilm. Assuredly, these evidences are only suggestive, the in-depth investigation of tolerance mechanisms are in progress.
Acknowledgements
This work was supported by grants from the National Outstanding Youth Foundation of China (Grant No. 21025625), the National High-Tech Research and Development Program of China (863) (Grant No. 2012AA021200), the National Key Technology R&D Program (2012BAI44G01), the National Natural Science Foundation of China (Grant No. 201390204, 21506090), Natural Science Foundation of Jiangsu (Grant No. BK20130929, BK20140940), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). The authors would like to thank Dr Liwen Mu at University of Akron for useful discussions regarding preparation of this manuscript.
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Footnote |
† These authors contributed equally to this work. |
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