Clostridia spp. are gram-positive bacteria that have important applications in industrial processes [1]. Indeed, in industry, solventogenic Clostridium spp. utilize carbon sources, such as glucose, xylose, mannose, and arabinose, to produce acetone, butanol, and ethanol during anaerobic metabolism. This fermentation process is called acetone-butanol-ethanol (ABE) fermentation [2, 3]. The main component, butanol, has the highest commercial value and is used as a multipurpose basic chemical raw material, high-quality renewable transportation fuel, and fuel additive [4]. Clostridium acetobutylicum, as the main strain used in ABE fermentation, expresses amylase and can be directly fermented with sugar and starchy raw materials without prior saccharification [1]. Owing to the unique sporulation and solvent production characteristics of C. acetobutylicum and its vital application value, many studies of C. acetobutylicum have been performed in the to evaluate the microbiological morphology, physiology, biochemistry, and molecular biology of the strain. Understanding the mechanisms through which Clostridium spp. adapt to environmental stress is essential for controlling these bacteria and taking advantages of its industrial applications [5].
The metabolic transition of C. acetobutylicum from the acidogenic period to the solventogenic period is an adaptive process resulting from the effects of acid toxicity on the bacterial cells [5]. During the solventogenic process, bacteria can metabolize the products accumulated during the acidogenic period through the reuse route, which causes the pH of the fermentation broth to rise, thereby partially detoxifying the acid [6]. Solvents produced during this process, particularly butanol, have major effects on the growth and metabolism of the bacteria and are therefore key factors limiting the fermentation yield [7, 8]. Therefore, butanol is considered the main toxic compound inhibiting the physiological activity of C. acetobutylicum.
Organic solvents first enter the cell membrane after coming in contact with the cells, and the phospholipid bilayer structure of the membrane is the main target of solvent toxicity [9]. The toxic effects of the solvent on the cell begin with binding of solvents to the phospholipid layer. After solvent accumulates on the cell membrane, membrane permeability to protons and ions increases [9], proton dynamics dissipate [10], energy conduction fails [11, 12], and intracellular pH control is suppressed, leading to penetration of intracellular macromolecules (such as RNA, phospholipids, and proteins) [11]. Moreover, solvent accumulation on the cell membrane also affects the activity of the mosaic protein by changing the physical and chemical properties of the membrane [13], such as alterations in the proton-K + pump [11, 12], and may alter the membrane structure, resulting in increased membrane fluidity [13], reduced microbial metabolism, and decreased growth. Thus, after the solvent enters the membrane, it disrupts the order of the membrane, weakening the function of the membrane as a penetration barrier and protein embedding platform. The increased permeability, fluidity, and disorder of the cell membrane make it difficult for microorganisms to resist the toxic effects of solvents, resulting in cell death.
Physiological studies of microorganisms have revealed that solvent toxicity is related to its logPo/w value [11]. The parameter logPo/w refers to the partition coefficient of a given solvent in equimolar octanol and water (indicating the hydrophobicity of the solvent) [14]. The greater the polarity, the smaller the logPo/w value and the greater the solvent toxicity [15]. The logPo/w values of common solvents range from 1 to 4.5, whereas that of butanol is 0.8 [16]. In addition, the solvent relies on its own hydrophobicity to enter and bind to different sites in the bilayer [14]. Binding of the solvent to different positions on the cell membrane can result in varying effects on membrane structure. For example, when a solvent is combined near the polar head, the effects of membrane disorder will be greater than that of the solvent deeply bound to the center of the phospholipid fat chain [17]. The binding positions of alkanols (e.g., butanol, octanol, dodecanol, and myristyl alcohol) on the cell membrane have been examined by Westerman et al. [18] and Pope et al. [19] by X-ray and nuclear magnetic resonance technology. The results indicated that amphiphilic molecules, such as alkanol, position the hydrated part of the molecule close to the polar head of the phospholipid molecule, whereas the fat chain portion is inserted between the fat chains of the phospholipid.
Among the four alkanols described above, butanol causes the most serious cell membrane lipid damage in the glycerol skeleton [18]. Indeed, Ezeji et al. [20] found that butanol inhibited the activity of bacteria by destroying the cell membrane and altering its functions, i.e., reducing the substance transfer capacity of the membrane. A series of reliable experimental results showed that there are two systems in C. acetobutylicum, i.e., a specific self-induced peptide transport system and a signal transduction system; the signal transduction system involves membrane receptor histidine kinase function [21]. Butanol toxicity also results in destruction of the membrane transport system (i.e., the phosphoenolpyruvate-carbohydrate phosphotransferase system), thereby preventing the transport and phosphorylation of glucose and inhibiting the transmembrane transport and assimilation of sugars, amino acids, and other nutrients [22].
In response to butanol toxicity on the cell membrane, a series of studies have been conducted to improve the ABE fermentation and solvent tolerance of C. acetobutylicum. With improvements in molecular biology technology, researchers have attempted metabolic engineering of C. acetobutylicum. For example, Xu et al. [23] knocked out CAC3319 (histidine kinase) in C. acetobutylicum and showed that the tolerance of the strain increased and that butanol production (18.2 g/L) and butanol yield (0.38 g/L/h) were both enhanced. Moreover, Borden et al. [24] screened the resistance genes of C. acetobutylicum by constructing a gene library and showed that recombinant strains carrying genes such as CAC0977, CAC1463, CAC1869, and CAC2495 were enriched. CAC1869 is a transcriptional regulator of the heterogeneous stress element, and its introduction into ATCC 824 increased the butanol tolerance of the recombinant strain by 90%. Mann et al. [25] overexpressed the genes groESL, grpE, and htpG in C. acetobutylicum and showed that 2.0% (v/v) butanol stress for 2 h improved butanol tolerance in engineered strains overexpressing these genes, with survival rates of 45% (groESL), 25% (grpE), and 56% (htpG). In addition to targeted genetic engineering methods, the genes and proteins related to butanol tolerance have been molecularly modified to enhance butanol tolerance and survival rates, thereby increasing butanol production. Furthermore, some induced mutations can also be used to obtain butanol-tolerant mutants. C. beijerinichii BA101, obtained by chemical mutagenesis in 1991, was used for this application [26]. C. acetobutylicum EA2018 strain was also chemical mutated to become an excellent butanol producer, showing a 10% increase in butanol production compared with the original strain; thus, this strain has been introduced in industrial applications in China [27]. Guo et al. [28] used C. beijerinichii NCIMB 8052 as the starting strain and utilized low-energy ion implantation (N+) for physical mutagenesis to obtain a mutant strain IB4 with high tolerance and high butanol production. Baer et al. obtained a solvent-tolerant mutant strain SA2 through mutagenesis screening and found that the saturation of cell membrane fatty acids was higher than that of the control strain C. acetobutylicum ATCC 824 under various culture conditions between 22 and 37 °C. Moreover, the fluidity of the cell membrane remained unchanged under different concentrations of butanol (0–1.5%, v/v) [11].
Compared with traditional mutagenesis technology, as an advanced and efficient mutagenesis technology, heavy ion beam irradiation results in high mutagenesis rates and yields relatively stable mutants, leading to its broad applications as a tool for creating new species. In terms of microbial breeding, approximately 30 genera have been subjected to heavy ion beam irradiation to produce novel strains [29–31]. Cadmium resistance of Arthrobacter mutants selected by carbon ion beam irradiation was increased by 2-fold [32], and Lu et al. [33] increased the fermentation rate of Saccharomyces cerevisiae subjected to carbon ion beam irradiation by 25%. Zhou et al. [34] used carbon ion beam irradiation to significantly improve the resistance of C. butyricum to butyric acid and the ability to produce butyric. Accordingly, many excellent microbial mutants with applications in industry and research have been bred using heavy ion beam irradiation mutagenesis.
C. acetobutylicum Y217 is a stable and high-yield resistant mutant selected by C. acetobutylicum ATCC 824 as the starting strain through carbon ion beam irradiation. Based on previous studies, Y217 maintains better cell integrity under butanol stress than ATCC 824, potentially because of its high butanol tolerance. In this study, we systematically analyzed and compared the cell morphology, cell surface hydrophobicity, membrane potential, membrane permeability, cell fatty acid composition, and fermentation kinetics of high-butanol tolerant mutant and wild-type strains under butanol stress in order to clarify the membrane physiology and metabolic mechanisms of C. acetobutylicum under butanol stress.