Experimental design and results for the central composite design.
In this study, cold plasma at atmospheric pressure, as a novel approach of bioprocess intensification, was used to induce yeast for the improvement of ethanol production. Response surface methodology (RSM) was used to optimize the discharge-associated parameters of cold plasma for the purpose of maximizing the ethanol yield achieved by cold plasma-treated S. cerevisiae. The resulting yield of ethanol reached to 0.48 g g−1 under optimized parameters of plasma exposure time of 1 min, power voltage of 26 V, and an exposed sample volume of 9 mL, which represented an increase of 33% over control. Compared with non-exposed cells, cells exposed with plasma for 1 min presented a notable increment in cytoplasmic free Ca2+, when these exposed cells showed the significant increase in membrane potential. At the same time, ATP level decreased by about 40%, resulting in about 60% reduction in NADH. Taken together, these data suggested that the mechanism that air cold plasma raised plasma membrane potential, which led to increases in cytosolic Ca2+ concentration. Furthermore, the cofactor metabolism, such as ATP and NADH, was subjected to regulation that was mediated by Ca2+, ultimately improving yeast productivity. This may have a underlying and broad utilization in enhancing bioconversion capability of microbe in the next few years.
- Saccharomyces cerevisiae
- cofactor metabolism
- bioprocess intensification
- cold plasma at atmospheric pressure
Bioethanol is currently being commercially produced as an alternative to petroleum-based transportation fuels, since it is clean, renewable, carbon-neutral and environmentally friendly [1, 2, 3].
Pretreatment technologies have been developed to intensify bioethanol production, including physical, chemical, biological and physicochemical technologies [5, 6, 7, 8, 9]. Furthermore, the methods to control the membrane permeability have also been established, such as microwave, electric field, oxidative stress [10, 11, 12]. However, these methods have several drawbacks. For example, the chemical methods could generate enormous amounts of hazardous waste, while physical methods are difficult to apply at large scales. It is therefore necessary to develop a novel approach to change cell membrane permeability for improved bioethanol yield.
Cold plasma at atmospheric air pressure has recently been regarded as a new and advantageous pretreatment technology result from its superior features of high efficiency, low energy consumption and environmentally friendly. Air cold plasma could present various biological effects on the microbes, such as activation effect, sterilization effect and mutagenesis effect, due to the changes in the concentration of reactive species caused by different parameters associated with the plasma discharge . Therefore, the discharge-associated parameters for improved ethanol yield need to be optimized. In this study, the response surface method (RSM) was performed to optimize experimental parameters that could cause the increase in the yield of ethanol generated by
The cell membrane is the first barrier that the substrate enters into the cytoplasm. Thus the improved membrane permeability would promote the glucose utilization and even ethanol release. The rapid consumption of glucose could disturb the cofactor metabolism (such as ATP, NADH et al.) and the re-distribution of carbon flux in glycolysis pathway . In addition, the open of ion channels is the one of mechanisms that the cell membrane permeability is improved. Especially, calcium ion channel administers the alterations of cytoplasm calcium ion concentration ([Ca2+]cyt). Ca2+, as a key secondary messenger, is importantly responsible for cell metabolism and activities of some categories of ATPase . As shown in Figure 1, a raise of [Ca2+]cyt can be result of improved inflow of extracellular Ca2+ by Cch1 protein/Mid1 protein (Cch1/Mid1 p) on cell membrane or as a result of outflow of vacuolar Ca2+ into the cytoplasm through vacuole membrane-located Yvc1 protein (Yvc1p) channel [17, 18, 19, 20]. Until now, little knowledge has been obtained on the relationship among air cold plasma, cell membrane permeability, cofactor metabolism and ethanol yield.
The object of this study was to achieve the maximum yield of ethanol by optimizing parameters associated with plasma discharge. Moreover, the mechanism of intensified yield of ethanol produced by
2. Results and discussion
2.1. Parameter optimization associated with plasma discharge for enhanced ethanol yield
2.1.1. Influence of plasma treatment time on ethanol yield
To achieve the maximum ethanol-yield, plasma treatment time was set at five different time intervals, from 1 to 5 min. Ethanol yield at 3 min reached to the maximum (0.45 g/g), and it presented an increase of 29% over the control (Figure 2). This indicated that a plasma treatment time of 3 min was appropriate for maximal ethanol production. Thus, 3-min treatment time was chosen as the treatment time for studying the influences of various power supply voltages and volumes of yeast suspension on ethanol yield. In our earlier research, the highest yield of 1,3-propanediol produced by
2.1.2. Influence of power supply voltage on ethanol yield
The influence of the power supply voltage in plasma treatment on ethanol yield is shown in Figure 3. Ethanol yield raised with raising power supply voltages, up to 0.42 g/g, then dropped with further increase in power supply voltage. The maximum yield of ethanol was achieved at 26 V.
It has been reported that charged particles in low-temperature plasma play a key role in the alterations of the outer structure of
2.1.3. Influence of treated suspension volume on ethanol yield
The influence of various sample volumes on the ethanol yield was studied for the maximal ethanol yield. As shown in Figure 4, a sample volume of 5 mL enhanced ethanol yield by 28% for the plasma pretreated cells over the control. Cell suspension also constitutes a dielectric layer. A larger suspension volume means that the thickness of the dielectric layer would increase in a Petri dish of 60-mm diameter, and any alteration about dielectric properties would also caused a alteration in discharge characteristics, especially for the power voltage . As a result, a sample suspension volume of 5 mL could show an impactful augment in ethanol yield.
2.1.4. Predictive response model
|Source||Sum of squares||Mean square|
The adequacy of the model was checked using analysis of variance (ANOVA), which was tested using Fisher’s statistical analysis . The Model F-value of 6.09 indicated model significance. Value of “Prob > F” less than 0.05 indicated that the model terms were remarkable, whereas values greater than 0.10 indicated no significance. ANOVA resulted in a value of 0.85 for the coefficient of determination (R2) and 0.71 for the adjusted coefficient of determination (R2adj). The R2adj value was close to 1, which indicated a high degree of correlation between the observed and predicted values .
2.1.5. Influence of various experimental parameters on ethanol yield
The influences of the independent parameters, including plasma treatment time, power supply voltage and induced-sample volume, on ethanol yield were analyzed by three dimensional response surface plots (Figure 5). Figure 5(a) presented the ethanol yield based on a combination of plasma treatment time and power supply voltage. The predicted ethanol yield showed to increases at 1 min and from 25 to 27 V. Figure 5 (b) presents the interaction between plasma treatment time and sample suspension volume on ethanol yield. The highest ethanol yield was achieved when 9-mL sample suspension was treated by dielectric barrier discharge (DBD) plasma for 1 min. The predicted ethanol yield of
2.1.6. Confirmation of optimum parameters
Optimum conditions of the parameters achieved from the above analysis were verified by carrying out flask fermentation with
|Groups||Biomass (g/L)||Glucose consumption (g/L)||Ethanol (g/L)||Ethanol yield (g/g)|
|Control group||5.4 ± 0.9||132.0 ± 8.3||47.5 ± 2.7||0.36 ± 0.02|
|Optimized group||6.7 ± 1.1||141.0 ± 10.8||67.5 ± 4.2||0.48 ± 0.03|
To enhance the concentration of ethanol, different methods have been used to improve the productivity of the correlative microorganism strains, including construction of genetic engineering strain , mutagenesis and breeding , as well as metabolism control by changing the osmotolerance of the external environment . Up to now, little study has been reported about the application of cold plasma at atmospheric pressure in intensifying ethanol yield of
The optimized parameters (1 min, 26 V, 9 mL) achieved by the central composite design experiment were different from the optimized parameters (3 min, 26 V, 5 mL) achieved by single-factor experiment. This may be due to the following reasons. Firstly, response surface methodology reflected the influences of interaction among the three parameters employed with the other parameter maintained at its respective zero level on ethanol yield. In this research, the dielectric layer became thick when the volume of the test sample was increased in an unchanged 60-mm-diameter Petri dish, causing an alteration in the power voltage. Therefore, the three parameters (plasma exposure time, test sample volume, power supply voltage) underwent a simultaneous alteration. Secondly, the plasma discharge device was directly laid in air at room temperature, and the discharge was affected by various environmental factors, such as air humidity and ambient temperature. Finally, experimental errors were observed during the operation. For example, the gap distance between electrodes was widened again and again for putting the sample on the bottom electrode before every experiment, and then the distance between electrodes was recovered.
2.2. Mechanism study about enhanced ethanol yield of
Saccharomyces cerevisiaewith cold plasma
2.2.1. Plasma membrane permeability
The alterations in membrane permeability exhibited by
2.2.2. Plasma membrane potential
The membrane potential was measured with the aid of the fluorescence probe Rh123 (Figure 7). The fluorescence intensity of Rh123 was positively correlated with plasma membrane potential. These data indicated that the plasma membrane permeability was increased (20%) when the samples were treated for 1 min, but was decreased when they were treated for 2–5 min. When the treated samples were cultured for 9 h, only the membrane potential of the sample treated for 1 min reduced relative to that of non-treated sample. Other exposure times gave various increases in membrane potential, among which 2 min exposure yielded the maximum increase (70%) compared with non-treated sample. In the case of 21-h fermentation, 4- and 5-min exposures gave remarkable improvements in membrane potential over non-treatment. These data seemed to show that cold air plasma discharge could either increase or decrease the plasma membrane potential of
2.2.3. Cytoplasmic calcium concentration
The intracellular calcium concentration of plasma-treated samples was detected using the fluorescence probe Fluo-3 AM (Figure 8). The calcium concentrations in the cytoplasm were improved with plasma treatment time, with 5 min treatment giving the maximal increase, about 36% more than the concentration measured in the non-treated cells. After 9 h of fermentation, cytoplasmic Ca2+ concentrations were significantly increased in the sample of 1- or 2-min plasma treatment over non-treatment of plasma, but in the samples from 3- to 5-min plasma treatment, Ca2+ concentrations were less compared with non-treatment of plasma.
2.2.4. Extracellular ATP concentration
The influence of plasma treatment on extracellular ATP concentration was most significant prior to fermentation (0 h) and at the 9-h stage of fermentation following plasma exposure (Figure 9). Prior to fermentation, some significant reductions in extracellular ATP concentration were measured when
2.2.5. Extracellular NADH concentration
Differences in extracellular NADH concentrations between non-treated and plasma-treated
In this research, we have proved that remarkable decrease in membrane permeability of live cells were distinct after the sample was treated by plasma for 1 min (Figure 6). At the 21-h periods of fermentation, the membrane permeability was increased showing that the effect of air cold plasma on membrane permeabilization was temporary and non-inheritable. This result was in accordance with the study of Yonson
Membrane potential is an important factor in cellular functions such as signaling and transport, which can eventually affect cell metabolism . An alteration in membrane potential can be positively detected by an alteration in fluorescence intensity of Rh123. When discharge plasma occurs over the solution surface, a variety of physical and/or chemical processes are activated. Many active species such as oxygen, hydrogen, hydroxyl and hydroperoxyl radicals are produced. These reactive species can diffuse in the surrounding liquid and induce the redistribution of charges on the inner and outer surfaces of the cell membrane, leading to an increase or reduction of membrane potential. Such change of the membrane potential would directly affect the plasma membrane permeability. After
The change of cell membrane potential could activate the voltage-dependent Cch1p channel, causing more influx of Ca2+ from the extracellular environment into the cytoplasm (Figure 1). Therefore, the calcium level in the cytoplasm of treated cells was enhanced after plasma treatment. Air cold plasma slightly improved the cytoplasmic calcium concentration of the sample following treatment for 1 min. This might result from the increase in plasma membrane potential (Figure 7 versus Figure 8, at 0-h culture), causing cell membrane hyperpolarization and opening of Ca2+ channels. But the opening of Ca2+ channels did not cause an increase in cell membrane permeability (Figure 6). This result suggests that the increment in cell membrane permeability might be controlled by more than one channel modulator.
The alteration trend of ATP concentration was different from the alteration tend in membrane permeability with plasma discharge. This shows that change of extracellular ATP concentration is a direct consequence of alterations in intracellular ATP. Before fermentation, the lower concentrations of ATP at 1 and 2 min plasma treatment might be due to 6.8 and 10% increments in calcium concentration, respectively. The increased calcium concentration promoted the hydrolysis of ATP to adenosine diphosphate (ADP) (Figure 9). A Ca2+ concentration gradient from 1 to 10 μM, could improve the cell function that regulates cell growth and metabolism to eventually enhance microbial productivity. However, the high concentrations of intracellular Ca2+ can induce cell injury or death [34, 35]. The higher concentrations of ATP in the samples treated by plasma for 3–5 min might be due to an inhibition of ATP hydrolysis caused by the higher cytoplasmic calcium concentration (Figures 8 and 9). In addition, any disturbance in environmental conditions would influence the activities of catabolic enzymes, thereby accelerating the accumulation of ATP or ADP . Air cold plasma might lead to the accumulation of ADP in the treated samples within 1–2 min of treatment, and of ATP in the treated samples within 3–5 min of treatment, as suggested by the data in Figure 9. The accumulation of ATP or ADP might have immediately affected the glycolysis rate , producing different ATP concentrations at the 9- or 21-h period of fermentation, depending on the plasma treatment time (Figure 9).
Air cold plasma produces different reactive species in the gas phase . These active species further react with water and produce a variety of biologically active reactive species (RS) in the liquid phase, including long-lifetime RS (ozone, hydrogen peroxide and nitrate ions) and short-lived RS (superoxide, hydroxyl radicals and singlet oxygen) . In our research, these reactive species could increase or decrease the cell membrane potential and open Ca2+ channels, consequently improving [Ca2+]cyt (Figures 7 and 8, at the beginning of culture). Ca2+ supplementations of 0.5 and 1.5 mM have been shown to induce the increment in ATPase activity . The enhanced ATPase activity would then promote the generation of proton motive force through hydrolysis of ATP [29, 39]. A reduction in the intracellular ATP level can result in the up-regulation of the activities of phosphofructokinase (PFK) and pyruvate kinase (PK) . This would accelerate the glycolytic flux and enhance the NADH level in the central metabolic pathway . At the same time, NADH-dependent alcohol dehydrogenase (ADH) activity might be improved, leading to up-control of the oxidation of NADH to NAD+ [40, 42] (Figure 1). Therefore, the NADH concentration obtained from 1 min treatment was reduced over the control because of the lower level of ATP (Figure 10 1 min versus Figure 9 1 min). The oxidation of NADH to NAD+ would lower the activity of NADH-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH), causing decreased glycerol production and ultimately causing more carbon flux from glycolysis being funneled to ethanol [42, 43, 44].
Experimental parameters associated with cold plasma discharge at atmospheric air pressure for enhancing ethanol yield of
Furthermore, the potential mechanism that air cold plasma alters the cofactor metabolism of
This work was financially supported by the National Natural Science Foundation of China (grant numbers 21246012, 21306015, and 21476032). The author thanks undergraduate students of X Wang, TT Liu and YQ Xiong for attending some works of experiments.
Conflict of interest
The author declares no financial or commercial conflict of interest.