A novel approach to regulate cell membrane permeability for ATP and NADH. formations in Saccharomyces cerevisiae induced by air cold plasma 1

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1 A novel approah to regulate ell membrane permeability for ATP and NADH formations in Saharomyes erevisiae indued by air old plasma 1 Xiaoyu DONG ( 董晓宇 ), Tingting LIU ( 刘婷婷 ), Yuqin XIONG ( 熊玉琴 ) Shool of Life Siene and Biotehnology, Dalian University, Dalian , China dongxiaoyu@dlu.edu.n Reeived 27 May 2016 Aepted for publiation 18 September 2016 Abstrat: Air old plasma has been used as a novel method for enhaning mirobial fermentation. The aim of this work was to explore the effet of plasma on the membrane permeability and the formations of ATP and NADH in Saharomyes erevisiae, so as to provide valuable information for plasma appliation in the large-sale fermentation industry. Suspensions of S. erevisiae ells were exposed to air old plasma for 0 min, 1 min, 2 min, 3 min, 4 min and 5 min, and then subjeted to various analyses prior to fermentation (0 h) and at the 9 h and 21 h stages of fermentation. Compared with non-exposed ells, ells exposed to plasma for 1 min exhibited a marked inrease in ytoplasmi free Ca 2+ onentration as a result of the signifiant inrease in membrane potential prior to fermentation. At the same time, ATP level in the ell suspension dereased by about 40%, resulting in about 60% redution in NADH prior to ulturing. However, the levels of ATP and NADH in the ulture at the 9 h and 21 h fermentation stages were different from the level at 0 h. Taken together, the results indiated that exposure of S. erevisiae to air old plasma ould inrease its ytoplasmi free Ca 2+ onentration by improving the ell membrane potential, onsequently leading to hanges in ATP and NADH levels. 1 Supported by National Natural Siene Foundation of China (Nos , )

2 Keywords: Saharomyes erevisiae, air old plasma disharge, membrane permeability, ofator PACS: j, s 1 Introdution Cell membranes not only gives rise to ell shape and provide protetion for the ell but they are also involved in metaboli proesses, suh as the transport of ions and nutrients, the storage and transmission of energy, as well as in ell signaling and ommuniation [1]. During biotransformation, the rates of nutrient uptake and metaboli produt release are greatly restrited by the ell membrane. Therefore, the prodution apaity of mirobial ell in bioatalysis and fermentation may be gravely ompromised [2]. It is, therefore, neessary to artifiially regulate ell membrane permeability to improve the yield of target produts in a biotransformation proess. Numerous pre-treatment tehnologies have been developed to ontrol the membrane permeability of mirobes. These tehnologies inlude osmoti pressure, oxidative stress, rare-earth ion exposure, eletri field and mirowave irradiation [3 7], and they work by altering the funtional properties of the ell membrane to promote the permeability and transport of ion and maromoleules. However, these methods have several drawbaks. For examples, hemial methods suh as osmoti pressure, oxidative stress, rare-earth ion exposure, generate enormous hazardous wastes, while physial methods, suh as eletri field and mirowave irradiation, are diffiult to apply in a large-sale implementation. Therefore, there is a need to develop a novel approah for altering the ell membrane permeability, one that is simple and has superior effiieny in addition to being an energy saving and environmental friendly proess. Suh a method would have a great benefit for the environment and a broad market prospet.

3 Air old plasma has reently been reognized as one of the novel and advantageous pre-treatment tehnologies beause of its high effiieny, low energy onsumption, and environmentally friendly features. In reent years, it has been widely used in sterilization and mutagenesis of industrial mirobes as a result of its physial and hemial properties [8 10]. In our earlier studies, we have reported that dieletri barrier disharge (DBD) plasma at air atmospheri pressure an improve the produtivity of 1,3-propanediol in Klebsiella pneumoniae by about 56% in bath fermentation [11]. The ell membrane permeability of the exposed ells is also markedly inreased throughout the fermentation [11]. Furthermore, ethanol yield by plasma-exposed Saharomyes erevisiae has a lose relationship with the plasma parameters, suh as plasma disharge time, power voltage and the volume of the ell suspension used in the exposure. Under the onditions of 1 min, 26 V and 9 ml, the final yield of ethanol is 0.48 g g -1, an inrease of 33% over ontrol [12]. Bakers' yeast, S. erevisiae, is widely used in the biologial prodution of ethanol. Inreasing the permeability of the yeast ell membrane would promote the rates of gluose uptake and utilization, thereby inreasing the ethanol yield in the fermentation. The gluose metaboli pathway of S. erevisiae during anaerobi fermentation is shown in figure 1. The triarboxyli aid yle (TCA) pathway does not operate as a yle in the mitohondria, as most of the earlier reports have desribed, but, rather, it ours as two branhes in the ytosol [13]. Cofators suh as adenosine triphosphate (ATP) and the redued form of niotinamide adenine dinuleotide (NADH) play important roles in the distribution and rate of metaboli fluxes in a metaboli pathway. ATP is obtained from glyolysis, and its inrease may enhane the growth rate of the ell or organism [14]. However, Larsson et al. reported a strong inverse proportional

4 relationship between intraellular ATP level and the rate of glyolysis; i.e., the lower the onentration of ATP, the higher the glyolysis rate [15]. The rapid onsumption of gluose ould lead to the aumulation of NADH. Most of the NADH produed during glyolysis is subsequently oxidized and used in the ethanol synthesis. Figure 1. Major pathway of gluose metabolism in S. erevisiae under anaerobi ondition. The alium ion is an important seondary messenger. Changes in ytosoli Ca 2+ onentration are assoiated with the regulation of an extensive variety of metaboli reations, suh as ell metabolism, ell signaling, membrane transport hannels and the ativities of some types of ATPases [16]. As shown in figure 1, a rise in Ca 2+ onentration in the ytoplasm an be the result of inreased extraellular Ca 2+ inflow through the ell membrane-loated Ch1 protein/mid1 protein (Ch1p/Mid1p) hannel or as a result of vauolar Ca 2+ outflow into the ytoplasm via the Yv1 protein (Yv1p) hannel on the vauole membrane [17 20]. The opening of ion hannel is one of the mehanisms by whih ell membrane permeability is enhaned. Up to now, little

5 information has been available on the impat of air old plasma on ell membrane permeability and the levels of ofators in yeast metabolism during ethanol fermentation. The aim of the present study was to explore the influene of air old plasma exposure time on the permeability of yeast ell membrane by measuring the hanges in ytoplasmi free Ca 2+ onentration after subjeting the ells to different plasma exposure times. In addition, hanges in the levels of the ofators, ATP and NADH, were also measured to investigate the effets of plasma exposure time on ofators' metabolism. This study aimed to provide useful information onerning the possible use of air old plasma to ontrol the metabolism of ofator in yeast ell through altering its membrane permeability. 2 Experiment 2.1 Miroorganism and growth ondition Yeast strain S. erevisiae CGMCC No was obtained from China General Mirobiologial Culture Colletion Center (CGMCC, Beijing, China). Yeast ells were grown in yeast extrat peptone dextrose (YPD) solid medium (yeast extrat, 10 g L -1 ; gluose, 20 g L -1 ; peptone 20 g L -1 ; agar 20 g L -1 ) for strain preservation and viable ount assay. YPD liquid medium was used for general ell propagation whereas YPD liquid medium ontaining 180 g L -1 gluose instead of 20 g L -1 was used for fermentation. 2.2 Preparation of pre-exposed sample Yeast CGMCC-6184 preserved in a refrigerator was revived on YPD agar medium at 28 o C for 36 h. After that, one loop of yeast ells was inoulated into 100 ml of YPD liquid medium and inubated on a rotary shaker at 200 rpm and 28 o C. The ells were harvested at the logarithmi-phase by entrifugation at g and 4 o C for 15 min. Finally, the ell pellet was

6 suspended in sterile YPD liquid medium to an optial density of 600 nm (OD 600) of 10 (about ells ml -1 ) for plasma exposure. 2.3 Cold plasma disharge proessing The old plasma disharge equipment and the main steps involved in the sample exposure proess using old plasma (Figure 2) were similar to those desribed in our earlier report [21]. The unexposed and exposed ell suspensions were immediately subjeted to further analyses as desribed below or to flask fermentation. Samples from a fermentation ulture were withdrawn at 9 h and 21 h for the same series of analyses. A more detailed desription an be found in a previous report [21]. (a) (b) hamber gas exhaust power amplifiers gas inlet eletrode ell suspension dieletri quartz glass YPD medium (Solid) ( YPD medium (Liquid) ( power generator resistor, 50Ω ( Fermentation medium ( D=3 mm Sample treated by plasma Figure 2. Shemati representation showing the essential features of an air old plasma disharge setup. (a) and the protool for enhaning membrane permeability in flask liquid ulture (b). 2.4 Determination of membrane permeabilization Membrane permeabilities of unexposed ells and ells exposed to plasma were determined at the 0 h, the 9 h and 21 h ulturing stages, as desribed previously [21], but with modifiation. Fluoresein diaetate (FDA) was added to the yeast suspension to a final onentration of 0.5

7 mg ml -1. After inubation in the dark at 37 o C for 5 min, the ells were olleted, washed twie in phosphate buffered saline (PBS) and then re-suspended in PBS to a density of ells ml -1. The fluoresene of the ells was measured with a fluoresene spetrophotometer (JASCO Japan C., Ltd.). Exitation and emission wavelengths were set at 269 nm and 517 nm, respetively. The fluoresene intensity of the sample was expressed as perentage relative to ontrol. 2.5 Determination of membrane potential The membrane potential of yeast ells was measured with a fluorospetrophotometer as desribed previously [21, 22]. Rhodamine123 (Rh123) was added to the ell suspension to a final onentration of 10 μg ml -1. After inubation in the dark at 37 o C for 30 min, the ells were olleted, washed twie and re-suspended in PBS to a onentration of ells ml -1. The ell suspension was subjeted to membrane potential measurement. Exitation and emission wavelengths were set at 244 nm and 486 nm, respetively. Fluoresene intensity was expressed as a perentage relative to the ontrol. 2.6 Determination of ytoplasmi Ca 2+ The pretreatment for Ca 2+ permeability with Fluo-3 AM as fluoresent probe was similar to the reported approah [21, 23]. Briefly, fluoresent probe Fluo-3 AM was added a suspension of yeast ells to a final onentration of 0.5 μmol ml -1. The ell suspension was ultured in dark at 37 o C for 30 min, followed by entrifugation at g for 15 min. The ells were washed and re-suspended in PBS. The amount of Ca 2+ in the ytoplasm was obtained from the fluoresene intensity measured with a fluorospetrophotometer at an exitation and emission wavelengths of 506 nm and 526 nm, respetively. The intensity was expressed as perentage relative to the ontrol.

8 2.7 Measurements of extraellular ATP and NADH ontents The ellular ontent of NADH and ATP may be released into the extraellular spae by ative exoytosis or diffusion aross membrane hannels under ondition of ell stress [24]. Therefore, the supernatants of the unexposed ell suspension and ell suspension that had been exposed to old plasma olleted after entrifugation were diretly used for ATP and NADH assays. ATP assay was performed with an ATP Colorimetri Assay Kit (NJJCBIO, China) aording to the manufaturer's instrution. NADH assay was performed using an Amplite TM Colorimetri Total niotinamide adenine (NAD) and NADH Assay Kit "Blue Color" (AAT Bioquest In, Sunnyvalve, CA, USA) aording to the manufaturer's instrution. 2.8 Protein assay The protein onentration of the above supernatants was measured using the Bradford assay [25]. 2.9 Data analysis All of the measurements were performed in tripliates. All of the data statistis, inluding means, standard errors, and signifiane omparison were alulated by Origin 7.0 software. Signifiant differenes between test samples and ontrols were onsidered at the P<0.05, P<0.005 or P<0.001 level. 3 Results 3.1 Plasma membrane permeability The hanges in membrane permeability exhibited by S. erevisiae ells following their exposure to plasma and subsequent ulturing under fermentation ondition are shown in Table 1. After exposure to plasma for 1 min, the membrane permeability dereased ompared to unexposed ells, but inreased when the exposure time was inreased from 2 min to 4 min, and then fell bak to the

9 level of unexposed ells when the exposure time was inreased to 5 min. The membrane permeability of the exposed ells reahed a maximum when the ells exposed for 4 min were ultured for 9 h, yielding a 1.2 fold inrease over that of unexposed ells. As for ells that were ultured for 21 h, signifiant inrease in membrane permeability only ourred for those that were derived from ells exposed to plasma for 1 min and 5 min. Table 1. Effet of plasma exposure on fluoresene intensity of FDA before and after fermentation Relative fluoresene intensity of FDA (%) Exposure Original period of Middle period of Final period of ulture ulture (0 h) ulture (9 h) (21 h) 0 min 100±0 100±0 100±0 1 min 87±3.1b 100± ±4.2b 2 min 115±3.8b 94± ±5.0 3 min 125± ±5.9a 101±4.6 4 min 127± ±9.3 97±4.5 5 min 103± ± ±5.9a Data were expressed as the mean±ses. a, b, and indiated P<0.05, P<0.005, and P<0.001, respetively. 3.2 Plasma membrane potential The membrane potential was deteted with the aid of the fluoresene probe Rh123 (Table 2). The fluoresene intensity of Rh123 positively orrelated with plasma membrane potential. These data showed that the plasma membrane potential was raised (20%) when the ells were exposed

10 for 1 min but was redued when they were exposed for 2 min to 5 min. When the exposed ells were ultured for 9 h, only the membrane potential of the ells derived from those exposed for 1-min dereased relatively to those derived from non-exposed ells. Other exposure times gave different inreases in membrane potentials, among whih 2 min exposure yielded the maximum inrease (70%) over non-exposed ells. In the ase of 21 h fermentation, 4 min and 5 min exposure gave signifiant inreases in membrane potential ompared to non-exposure. These data seemed to indiate that old air plasma disharge ould either enhane or redue plasma membrane potential of S. erevisiae ells. Table 2. Effet of plasma exposure on fluoresene intensity of Rh123 before and after fermentation Relative fluoresene intensity of Rh123 (%) Exposure Original period of ulture Middle period of ulture Final period of ulture (0 h) (9 h) (21 h) 0 min 100±0 100±0 100±0 1 min 120±5.3b 93±3.0a 106±4.0 2 min 76± ±6.3 96±4.9 3 min 67± ±5.9b 108±5.3 4 min 74± ± ±6.5b 5 min 86±3.9b 105± ±5.0a Data were expressed as the mean±ses. a, b, and indiated P<0.05, P<0.005, and P<0.001, respetively. 3.3 Cytoplasmi alium ontent

11 The intra-ellular alium onentration of plasma-exposed ells was measured with the fluoresene probe fluo-3 AM (Table 3). The alium onentration in the ytoplasm inreased with plasma exposure time, with 5 min exposure gave the highest inrease, about 36% more than the onentration deteted in the non-exposed ells. After 9 h of fermentation, ytoplasmi Ca 2+ onentrations were markedly raised in the ase of 1 min and 2 min plasma exposure ompared to no plasma exposure, but in the ase of 3 5 min plasma exposure, ytoplasmi Ca 2+ onentrations were lower ompared to no plasma exposure. Table 3. Effet of plasma exposure on fluoresene intensity of Fluo-3 AM before and after fermentation Relative fluoresene intensity of Fluo-3 AM (%) Exposure Original period of ulture Middle period of ulture Final period of ulture (0 h) (9 h) (21 h) 0 min 100±0 100±0 100±0 1 min 107± ± ±4.0 2 min 110±4.0b 108±2.9b 102±4.6 3 min 115±5.0b 96±3.0 98±3.7 4 min 117±5.3b 59±2.4 95±3.6 5 min 136±4.8 88±2.0b 102±5.2 Data were expressed as the mean±ses. a, b, and indiated P<0.05, P<0.005, and P<0.001, respetively. 3.4 Extraellular ATP ontent The effet of plasma exposure on extraellular ATP onentration was most dramati prior to

12 fermentation (0 h) and at the 9 h stage of fermentation following plasma exposure (Figure 3). Prior to fermentation, some signifiant dereases in extraellular ATP onentration were deteted when S. erevisiae ells were exposed to plasma for 1 min and 2 min, but the signifiant inreases in ATP ontent ourred when the ells were exposed to plasma for 3 5 min ompared to non-exposed ells. At the 21 h stage of fermentation, however, extraellular ATP ontent for 1 min and 5 min exposure appeared to be somewhat lower than that of non-exposed ells. The result, therefore, indiated that plasma exposure might alter the onentration of extraellular ATP either immediately after treatment or when the treated ells were allowed to propagate for a moderate period of time under normal ulturing onditions. ATP ( mol mg -1 ) b b a b Time (h) a b b 0-min 1-min 2-min 3-min 4-min 5-min b Figure 3. Effet of plasma exposure on extraellular ATP before and after fermentation. Data were expressed as the means±ses. a, b, and indiated P<0.05, P<0.005, and P<0.001, respetively.

13 3.5 Extraellular NADH ontent Differenes in extraellular NADH onentration between non-exposed and plasma-exposed S. erevisiase ells were less uniform for all the three stages of testing. The differenes were more pronouned between non-exposed ells and exposed ells prior to fermentation or at the 21 h stage of fermentation (Figure 4). Prior to fermentation, 1-min treatment indued a derease of 60%, but 2 min and 3 min treatments aused 0.8 fold and 1.8 fold inreases, respetively, in extraellular NADH onentration. At the 9 h fermentation stage, extraellular NADH onentrations of exposed ells were either similar to or signifiantly lower than that of non-exposed ells. However, ells that were exposed to plasma for 1 min exhibited signifiantly higher extraellular NADH onentration than non-exposed ells at the 21 h fermentation stage although it remained muh lower than that of non-exposed ells in the other two stages (0 h and 9 h). In addition, 5 min exposure also showed signifiantly higher extraellular NADH onentration than non-exposed ells at the 21 h fermentation stage. Taken together, these results suggested that plasma exposure ould alter the extraellular onentration of NADH, either immediately after treatment or in subsequent fermentation, depending on the exposure time. NADH (mmol mg -1 ) Time (h) b 0-min 1-min 2-min 3-min 4- min 5-min b

14 Figure 4. Effet of plasma exposure on extraellular NADH before and after fermentation. Data were expressed as the mean±ses. a, b, and indiated P<0.05, P<0.005, and P<0.001, respetively. 4 Disussion In this study we have demonstrated that notieable inreases in membrane permeability of live ells were evident after the ells were exposed to plasma exposure for 1 min (Table 1). At the 9 h and 21 h stages of fermentation, the membrane permeability was redued, indiating that the influene of air old plasma on membrane permeabilization was temporary and non-inheritable. Our result was in aordane with the finding of Yonson et al. who reported that ell membrane permeability is temporarily enhaned by a miniature atmospheri pressure glow disharge plasma torh [26]. Membrane potential is a key fator in ellular funtions suh as signaling and transport, whih an ultimately affet ell metabolism [27]. A hange in membrane potential an be positively deteted by a hange in fluoresene intensity of an appropriate fluorophore, suh as Rh123. When disharge plasma takes plae over the solution surfae, a variety of physial and/or hemial proesses are initiated. Lots of ative speies suh as oxygen radial, hydrogen radial, hydroxyl radial and hydroperoxyl radial are generated. These reative speies an diffuse in the surrounding liquid and indue the redistribution of harges of the inner and outer surfae of the ell membrane, resulting in inrement or redution of membrane potential. Suh alteration of the membrane potential would diretly influene the plasma membrane permeability. When S. erevisiae ells were exposed to air old plasma, the hange in the membrane potential immediately after treatment ontrasted with the hange in membrane penetrability (Table 2 versus

15 Table 1). The ell membrane was depolarized due to the lowered potential, ultimately enhaning the permeability of the membrane. More inorgani and organi ions an then pass freely through the ell membrane as a result of this enhanement in permeability [28]. After the 9 h and 21 h stages of fermentation, the membrane potential inrement aused membrane hyperpolarization, and onsequently improving the membrane permeability. The alteration of ell membrane potential ould ativate the voltage-dependent Ch1p hannel, leading to more influx of Ca 2+ from the extraellular environment into the ytoplasm (Figure 1). Thus the alium level in the ytoplasm of exposed ells was improved after plasma exposure. Air old plasma slightly inreased ytoplasmi alium onentration of the ells following 1-min exposure time. This might result from the inrease in plasma membrane potential (Table 2 versus Table 3, at 0 h ulture), leading to ell membrane hyperpolarization and opening of Ca 2+ hannel. However, the opening of Ca 2+ hannel did not lead to an inrement in ell membrane permeability (Table 1). This result suggested that the inrease in ell membrane permeability might be governed by more than one hannel modulator. The trend assoiated with the hanges in ATP ontent in the ase of plasma exposure was different from that assoiated with the hanges in membrane permeability with respet to exposure time. This indiated that alteration of extraellular ATP ontent was a diret onsequene of hanges in intraellular ATP. Prior to fermentation, the lower levels of ATP at 1 and 2 min plasma exposure might be due to their 6.8% and 10% inreases in alium onentration, respetively. The inreased alium onentration promoted the hydrolysis of ATP to adenosine diphosphate (ADP) (Figure 3). A Ca 2+ onentration gradient from 1 to 10 μm, ould improve the ell funtion that regulates ell growth and metabolism to ultimately enhane mirobial produtivity. However,

16 the high levels of intraellular Ca 2+ an ause ell injury or death [29,30]. The higher levels of ATP in the ells exposed to plasma for 3 5 min might be due to the inhibition of ATP hydrolysis aused by higher ytoplasmi alium onentration (Table 3 and figure 3). In addition, any disturbane in environmental onditions would affet the ativities of ataboli enzymes, thereby aelerating the aumulation of ATP or ADP [30]. Air old plasma might ause the aumulation of ADP in the exposed ells within 1 2 min exposure, and of ATP in the exposed ells within 3 5 min exposure, as suggested by the data in Figure 3. The aumulation of ATP or ADP might have instantly influened the glyolysis rate [31], produing yet different ATP onentrations at the 9 h or 21 h stage of fermentation, depending on the plasma exposure time (Figure 3). Air old plasma generates various reative speies in the gas phase [32]. These ative speies further reat with water and produe a variety of biologially ative reative speies (RS) in the liquid phase, inluding long lifetime RS (hydrogen peroxide, ozone, and nitrate ion) and short-lived RS (hydroxyl radial, superoxide and singlet oxygen) [33]. In our study, these reative speies ould raise or lower the ell membrane potential and ativate Ca 2+ hannels, onsequently inreasing [Ca 2+ ]yt (Tables 2 and 3, at the beginning of ulture). Ca 2+ supplementations of 0.5 mm and 1.5 mm have been shown to indue the inrement of ATPase ativity [34]. The enhaned ATPase ativity would then promote the generation of proton motive fore through hydrolysis of ATP [34,35]. Dereasing intraellular ATP level an result in the up-regulation of the ativities of phosphofrutokinase (PFK) and pyruvate kinase (PK) [36]. This would aelerate the glyolyti flux and enhane the NADH level in the entral metaboli pathway [15]. At the same time, NADH-dependent alohol dehydrogenase (ADH) ativity might inrease, ausing an up-regulation of aetaldehyde to ethanol onversion, aompanied by the oxidation of NADH to

17 NAD + [36,37] (Figure 1). Therefore, the NADH level obtained from 1 min exposure was dereased ompared with ontrol beause of lower level of ATP (Figure 4 1 min versus figure 3 1 min). The oxidation of NADH to NAD + would lower the ativity of NADH-dependent glyeraldehyde-3-phosphate dehydrogenase (GAPDH), leading to redued glyerol prodution and eventually resulting in more arbon flux from glyolysis being funneled to ethanol [12,37,38]. 5 Conlusion The potential mehanism with regard to air old plasma-indued hanges in ofator metabolism of S. erevisiae was investigated in this study through analyzing the alterations in plasma membrane potential, ytoplasmi alium onentration, and the two ofators, ATP and NADH. Cells exposed to plasma for 1 min presented a remarkable inrease in plasma membrane potential, whereas ells exposed to plasma for 2 5 min showed notable dereases in plasma membrane potential. Furthermore, the onentrations of Ca 2+ for 1 5 min exposures were notieably inreased before the start of the ulture ompared with those of unexposed ells. The 7.0% inrease of alium onentration aused signifiant dereases in ATP (40%) and NADH (60%) at the 1 min plasma exposure. At 9 h fermentation, ATP ontent in ells derived from 1 min of plasma exposure inreased by 72% whereas NADH ontent dereased by 88% relative to the ontrol. Taken together, the mehanism by whih plasma indued hanges in ofators level in S. erevisiae appeared to be through raising the plasma membrane potential, whih then led to inreases in ytosoli free Ca 2+ onentrations within the ells, ultimately improving mirobial produtivity. This may have an underlying and broad appliation in enhaning the bioonversion apability of mirobes in the future. Referenes

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