ORIGINAL ARTICLE

Physiological comparison of cells with high and low alcoholdehydrogenase activities in bacterial populations consumingethanol

Volodymyr Ivanov & Saeid Rezaeinejad &

Olena Stabnikova

Received: 8 May 2014 /Accepted: 15 July 2014 /Published online: 3 August 2014# Springer-Verlag Berlin Heidelberg and the University of Milan 2014

Abstract Measuring the physiological heterogeneity of nat-ural and industrial microbial populations is essential to study-ing, modelling and monitoring of microbial populations. Itwas discovered that populations of Escherichia coli andBacillus megaterium growing in medium with ethanol as anexternal source of energy have two actively respiring butphysiologically different subpopulations. Cells of one subpop-ulation have negligibly low alcohol dehydrogenase (ADH)activity (ADH-L cells) and cells of the other have high ADHactivity (ADH-H cells). The subpopulation of ADH-H bacte-rial cells was measured using 10 min incubations of cells in a1% solution of allyl alcohol for fast selective killing of cellswith high activity of ADH and flow cytometry detection ofdead cells after this incubation. The content of ADH-H cellsduring exponential phase of batch culture varied from 9 % to90 % and lowered to zero for a few hours during starvation ofthe population. ADH-L cells are actively respiring cells andnot depolarized cells. The simultaneous presence of ADH-Land ADH-H cells growing in the medium with ethanol can beexplained by the fact that ADH-H cells oxidize actively ex-ternal ethanol whereas ADH-L cells oxidize only intracellular

storage carbohydrates. The method for enumeration of cellswith high ADH activity can be used to monitor the heteroge-neity of bacterial populations consuming ethanol as a solesource of carbon and energy.

Keywords Flow cytometry . Subpopulations . Alcoholdehydrogenase . Escherichia coli . Bacillus megaterium

Introduction

A bacterial population always contains cells at different levelsof physiological and biochemical activity due to either physio-logical changes of the cell during its life cycle (Ivanov 2010;Lidstrom and Konopka 2010; Lenz and Sogaard-Andersen2011; Ivanov et al. 2013; Magdanova and Golyasnaya 2013)or the spatial heterogeneity and existence of micro-gradients ofnutrients and metabolites in a population (Ivanov et al. 2008;Stewart and Franklin 2008; Muller et al. 2010). Therefore, sub-populations of cells with different values of physiological orbiochemical parameters are detected in isogenic growing cellpopulations (Amor et al. 2002; Breeuwer and Abee 2004;Duhamel et al. 2008; Muller and Nebe-von-Caron 2010;Rezaeinejad and Ivanov 2011; Ivanov et al. 2013).

There is also diversity in cell survivability among bacterialcells of one population upon exposure to stress, starvation, orantibiotics (Booth 2002; Dhar and McKinney 2007). This isespecially important for medical, environmental, and foodmicrobiology because a small fraction of a population cansurvive a treatment that kills the majority of the population.Differences in the survivability of bacterial cells is probablydue to the presence of some portion of cells with low or zerometabolic activity existing in an abiotic form of the life cyclesuch as spores, cysts, or dormant cells (Lennon and Jones2011). These persistent forms of bacterial cells form a smallfraction of the exponentially growing population but this

V. Ivanov (*)Department of Civil, Construction & Environmental Engineering,Iowa State University, Ames, IA 50011, USAe-mail: vivanov@iastate.edu

V. Ivanov : S. RezaeinejadDepartment of Civil and Environmental Engineering, NanyangTechnological University, Singapore 639798, Singapore

O. StabnikovaDepartment of Biotechnology and Microbiology, NationalUniversity of Food Technologies, 68 Volodymyrskaya Str.,Kiev 01601, Ukraine

Present Address:S. RezaeinejadBIOKUBE, Centervej syd 5, 4733, Tappernøje, Denmark

Ann Microbiol (2015) 65:1007–1016DOI 10.1007/s13213-014-0945-5

fraction can be increased by exposure of cells to nutrientstarvation or physicochemical stresses (Oliver 2010; Woodet al. 2013).

Ethanol is produced on a large scale by chemical andbiotechnological industries as a chemical commodity and abiofuel. It is used in biotechnology for cultivation of aceticac id bac t e r i a (Yakush i and Matsush i t a 2010) ,polyhydroxybutyrate-producing bacteria (Bhattacharyyaet al. 2014), production of single-cell protein (Nasseri et al.2011) and bacterial exopolysaccharides (Pirog et al. 2003).The key enzyme for the oxidation of ethanol is alcohol dehy-drogenase (ADH, EC 1.1.1.1). A solution of allyl alcohol isoften used to kill cells with genotypically high ADH activityduring batch cultivation. This method is used for selection ofmicrobial strains without ADH activity (adh−mutant) becausecells with high cytoplasmic ADH activity oxidize not onlyethanol but also allyl alcohol, thus producing toxic allyl alde-hyde, which kills these cells (Zhu et al. 2011; Plapp et al.2013; Scheel and Lütke-Eversloh 2013). Here, we used allylalcohol to kill cells with high ADH activity after a short-termincubation (less than one generation time) of cells in a solutionwith a low concentration of allyl alcohol, with the aim ofenumerating physiologically different cells with phenotypi-cally high (ADH-H cells) and low (ADH-L cells) ADHactivity.

The aim of this research was to discriminate and enumeratecells with high and low ADH activity in bacterial populationsgrowing in media with ethanol as a source of energy.

Materials and methods

Microorganisms and their cultivation

The strains Escherichia coli DSM 1329 and Bacillusmegaterium DSM 32 were obtained from the GermanCollection of Microorganisms and Cell Cultures (DSMZ).Cells were grown aseptically and aerobically in 500 mLErlenmeyer flasks with cotton plugs on a shaker at 200 rpmat 37 °C in 100 mL of the following medium (g/L in tapwater): NH4NO3, 2 g; KH2PO4, 3 g; K2HPO4, 7 g; MgCl2,0.1 g, with addition of either 0.5 % (v/v) ethanol or 1 % (w/v)glucose as a sole source of carbon and energy.

Phases of batch cultures

Growth was monitored by measuring optical density of thecell suspension at 600 nm using a spectrophotometer (modelDU 640B, Beckman Coulter, Brea, CA). Fast exponentialgrowth of the E. coli population was observed during theperiod from 1 h to 4 h of cultivation, slower exponentialgrowth was from 4 h to 14 h, and the stationary phase wasseen after 22 h of batch cultivation. Fast exponential growth of

the B. megaterium population was observed from 18 h to 30 hof batch cultivation, with stationary phase after 50 h of batchcultivation.

Respiration rate measurement

CO2 production rate was measured using a respirometer(Columbus Oxymax ER, Columbus, OH) connected to a50 mL sample of bacterial cells placed in 100 mL DURAN®cylindrical culture bottles (Schott, Elmsford, NY) in the shak-ing incubator at 37 °C and 200 rpm. The bottle was connectedto the respirometer and aerated continuously with an air flowof 100 mL/min so that the air retention time in the gas spacewas 0.5 min.

Starvation experiments

For experiments on starvation, 10 mL cells was washed twicewith phosphate buffered saline (PBS) (pH 7.2) using centri-fugation for 10 min at 3,400 g. The pellet was re-suspended in10 mL PBS and incubated on the shaker for 2.5 h. Sampleswere taken regularly for the 10-min treatment in 1 % (v/v)solution of allyl alcohol followed by flow cytometry.

Discrimination of ADH-H and ADH-L cells

Cells oxidizing external ethanol have high cytoplasmic ADHalcohol dehydrogenase (ADH, EC 1.1.1.1) activity. This en-zyme can also oxidize allyl alcohol thus producing the intra-cellular toxic acrolein (allyl aldehyde). Therefore, we incubat-ed cells in a 0.5–1.0 % (v/v) solution of allyl alcohol to allowthe physiological discrimination of cells with high ADH ac-tivity. A similar approach has been used in genetics for selec-tion of adh− mutants (Zhu et al. 2011; Plapp et al. 2013;Scheel and Lütke-Eversloh 2013).

After 10 min incubation in a 1 % (v/v) solution of allylalcohol, dead ADH-H cells were detected using flow cytom-etry after staining the cells with propidium iodide (PI). PI isused commonly as an indicator of dead cells (Shapiro 2000,2003; Hoefel et al. 2003; Berney et al. 2007; Doherty et al.2010; Muller and Nebe-von-Caron 2010) because the stainingof chromosomal DNA and part of rRNA with PI throughintercalation is possible only when a cell is dead and the cellmembrane has lost its integrity and become permeable formolecules of PI. A 1-mL aliquot of growing cell culture wasmixed with 10 μL allyl alcohol [final concentration 1 %(v/v) = 147 mM], in a 10 mL tube and incubated on the shakerat 200 rpm at 25 °C for 10 min. As a control, 200 μL allylalcohol was added (final concentration of allyl alcohol 20 %v/v) to kill all cells in the population. After incubation, cellswere washed twice with membrane-filtrated PBS (pH 7.2)using centrifugation for 10 min at 3,400 g. The pellet wasre-suspended in PBS and stained with 1 μL of the mixture

1008 Ann Microbiol (2015) 65:1007–1016

from the LIVE/DEAD BacLight™ viability kit, containingmembrane-permeable nucleic acids stain SYTO9 (3.34 mM),and PI (20 mM) (Molecular Probes, Invitrogen, Eugene, OR).Staining was performed in the dark for 20 min at 25 °C andcells were washed with PBS twice using centrifugation at3,400 g for 10 min and then re-suspended in 1 mL PBS forflow cytometry. Bacterial cells were detected by either for-ward scattering of light or by green fluorescence of SYTO9stain (Molecular Probes) for nucleic acids. The flow cytome-try gate for double stained dead cells, shown as R1 of dot plotin Fig. 2, was selected using cells killed in the 20% (v/v)solution of allyl alcohol.

Discrimination of respiring from non-respiring cells

The means of determining individual cell respiration ratethrough integrated activity of numerous intracellular dehydro-genases was via incubation of cells in a solution of 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), which is reduced by avariety of dehydrogenases to form a water-insoluble intracel-lular formazan having red fluorescence (Shapiro 2000, 2003;Creach et al. 2003; Czechowska et al. 2008). CTC is oftenused as an indicator of cell respiratory activity in combinationwith flow cytometry (Kaprelyants and Kell 1993; Lopez-Amoros et al. 1995; Shapiro 2000, 2003). In our experiments,an aliquot of 100 μL CTC (Polyscience, Warrington, PA)stock solution (50 mM) was added to a test tube containing900 μL sample and incubated for 1 h at 30 °C under agitationat 200 rpm. The final concentration of CTC was 5 mM. Redfluorescence of cells increased when formazan—the productof CTC reduction by dehydrogenases—accumulated in thecells. As a control, 200 μL ally alcohol was added [finalconcentration of allyl alcohol 20% (v/v)] to kill all cells inthe population, making them non-respiring. The flow cytom-etry gates for CTC-stained non-respiring cells are R1 of thedot plot in Fig. 3, and Q4 for CTC and DiBAC4(3) doublestained non-respiring cells, which were selected using cellskilled in the 20% (v/v) solution of allyl alcohol, are shown inFig. 6.

Discrimination of membrane-depolarized (dead) cells

Cells were also stained with bis-(1,3-dibutylbarbituric acid)trimethine oxonol, DiBAC4(3) (Molecular Probes, Invitrogen)at a final concentration 1 μM to detect the presence or absenceof cell membrane potential. Green fluorescence of the cellappears when the cell membrane is depolarized andDiBAC4(3) can penetrate and accumulate into the cell. Theconcentration of stock solution of DiBAC4(3) in dimethylsulfoxide (DMSO) was 250 μM. It was stored at −20°C. Analiquot of 4 μL of this stock solution was added to a test tubecontaining 996 μL cell suspension, then incubated at 37°C for20 min and washed twice by centrifugation at 3,398 g for

5 min, re-suspended in 1 mL PBS and analyzed immediatelyby flow cytometry (Shapiro 2003). As a control, 200 μL allyalcohol was added [final concentration of allyl alcohol 20%(v/v)] to kill all cells in the population making them non-polarized. The flow cytometry gate for double stained CTCand DiBAC4(3) depolarized cells, which were selected usingcells killed in 20% (v/v) solution of allyl alcohol, is Q4 inFig. 6.

Flow cytometry analysis of cell populations

Flow cytometry analysis was performed using aFACSCaliburTM flow cytometer (BD Biosciences, San Jose,CA) with CELLQuest TM software for data acquisition. A totalof 50,000 cells were counted, based on 488 nm excitation by a15 mWair cooled, blue argon ion laser. Green fluorescence ofSYTO9 was measured using a short pass filter 530 ± 15 nm,and red fluorescence of PI was measured using a band passfilter 620 ± 10 nm. The sheath flow rate was 10 mL/min. Thesample analysis rate was below 1,000 events/s. Flow cytom-etry data were analyzed using FlowJo V.7.2.5 software (http://www.flowjo.com). Digital images were produced using aFluoview300 confocal laser scanning microscope (Olympus,Tokyo, Japan). Red fluorescence of PI was excited by a10 mW or 40 mW argon ion laser at 488 nm, separated witha 570 nm splitting filter and detected in channel 1 with alongpass filter 510 nm (or band pass filter 525–575 nm) andin channel 2 with the bandpass filter at 580–640 nm (or 595–645 nm). The following gating strategy was used for all datafiles: cells treated with a 20 % (v/v) solution of allyl alcoholformed either the region R1 in Fig. 2 to detect dead cells byred fluorescence of PI, the region R1 in Figs. 3 and 5 to detectcells that were not reducing CTC, or quadrant Q4 in Fig. 6 todetect cells that were depolarized.

Results

Effect on cell respiration rate of incubation in 1 % solutionof allyl alcohol

The respiration rate of bacterial cells growing in medium withglucose as a sole source of carbon and energy did not changeafter a 10-min treatment in 1 % (v/v) allyl alcohol, and slightlydiminished after a 1-h treatment (Fig. 1). Meanwhile, therespiration rate of bacterial cells growing in medium withethanol as a sole source of carbon and energy was quicklydiminished after addition of 1 % (v/v, final concentration) ofallyl alcohol but after 10 min of treatment the respiration ratestabilized for more than 1 h at the same level (Fig. 1). Thisbehavior allowed us to use this treatment to discriminate cells

Ann Microbiol (2015) 65:1007–1016 1009

with high and negligibly low ADH activity, ADH-H andADH-L cells, respectively.

Effect on cell survival their incubation in 1 % solution of allylalcohol

Bacterial cells of populations of E. coli DSMZ 1329 andB. megaterium DSMZ 32, which grew exponentially in me-dium with glucose as a source of energy, were not sensitive toa 10-min treatment in a 1 % (v/v) solution of allyl alcohol(data not shown). Thus, all cells in populations that grewexponentially in media with glucose as a source of energywere ADH-L cells, i.e., cells with negligibly low ADH activ-ity. Meanwhile, in populations that grew exponentially inmedium with ethanol as a sole source of carbon and energy,some cells of E. coli DSMZ 1329 and B. megaterium DSMZ32were killed after a 10-min incubation in a 1% (v/v) solutionof allyl alcohol (ADH-H cells) but some cells remained aliveafter this treatment (ADH-L cells). Thus, populations growingin media with ethanol as a sole external source of carbon andenergy have two physiologically different subpopulations ofcells: one with negligibly low ADH activity (ADH-L cells)and another with high ADH activity (ADH-H cells). Anexample of a typical flow cytometry dot plot distribu-tion of ADH-L and ADH-L cells in an exponentiallygrowing population of E. coli DSM 1329 is shown inFig. 2. The flow cytometry dot plots show that about95% of cells were alive (Fig. 2a), but after 10 minincubation in a 1 % (v/v) solution of allyl alcohol, thecontent of living cells diminished to 79 % (Fig. 2b).The difference between the cell make up in the regionR2 before and after incubation in a 1 % solution ofallyl alcohol is due to the number of ADH-H cells. TheADH-L cell content is shown in region R2 of the dotplot in Fig. 2a–c.

Cells that died after incubation in a 1 % (v/v) solution ofallyl alcohol for 10 min can also detected not only by PIstaining but also by CTC staining (Fig. 3). ADH-H andADH-L cells of E. coli were also clearly discriminated oncolor images produced using dual PI + SYTO9 staining andconfocal laser scanning fluorescence microscope or fluores-cence microscope (color images not shown here).

The population of E. coli DSM 1329 cells contained asmall quantity of dead cells [3.4 ± 0.7 % (average ± standarddeviation), three independent measurements] during the ex-ponential phase of batch culture. The content of ADH-H cellsin the same samples taken during exponential growth of thepopulation varied from 9.4 % to 21.2 %. Average content ofADH-H cells ± standard deviation for three different samplestaken in the growth phase of batch culture was 15.8 ± 4.9%;however, the content of ADH-H cells in samples taken fromthe population of E. coli DSM 1329 growing exponentiallydiminished rapidly from 21 % to 1 % during 2.5 h of starva-tion (Fig. 4).

The fraction of ADH-H cells in the samples taken duringthe phase of fast exponential growth, between 18 and 30 h ofbatch culture of B. megateriumDSM 32, increased from 15%to 90%.However, in the sample, taken from themiddle of thisphase, the fraction of ADH-H cells of B. megaterium droppedfrom 28 % to 3 % during 3 h of starvation (Fig. 4).

Respiring and live non-respiring cells in the populationof E. coli DSM 1329 growing exponentially

The rate of CO2 production in batch culture of E. coli wasalmost constant for the period from 4 h to 24 h of batch cultureand did not correlate with the specific growth rate of thepopulation. The average content ± standard deviation of ac-tively respiring cells (CTC+ cells) in samples of the E. coliDSM 1329 population taken at the maximum rate of

0

20

40

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120

0 10 20 30 40 50 60 70 80 90 100

Time of incubation, min

Res

pir

atio

n r

ate

in t

he

sam

ple

, %

of

max

imumFig. 1 Effect of a 1 % (v/v, final

concentration) solution of allylalcohol on the continuouslymeasured respiration rate ofBacillus megaterium DSM 32.Arrow shows an addition of allylalcohol solution, solid linerepresents respiration rate afteraddition of allyl alcohol, dottedline shows respiration rate incontrol without addition of allylalcohol

1010 Ann Microbiol (2015) 65:1007–1016

exponential growth (3 h of cultivation), at slower exponentialgrowth (7 h of cultivation) and absence of growth (23 h ofbatch cultivation, see Fig. 1) were 77 ± 3%, 81 ± 3%, and

94 ± 3%, respectively. An example of the determination withthe related gates is shown in Fig. 5a–d. The growth rates atthese points of batch culture were 0.91, 0.20 and 0.01 1/h,respectively, but the specific respiration rates at the samepoints were 15.7, 10.0 and 2.8 mg CO2 h

−1 unit of OD600−1,

respectively. So, the content of CTC+ cells did not correlatewith either the specific growth rate or the specific respirationrate during exponential growth of the bacterial population.

During the exponential phase of batch cultivation of theE. coli population, from 17% to 20% of cells were not stained

a

c

b

Fig. 2 Flow cytometry dot plots of Escherichia coli DSM 1329 cellsbefore (a) and after a 10-min incubation with a 1 % (v/v) solution of allylalcohol (b) of samples taken after 24 h cultivation in medium containing0.5 % (v/v) ethanol. c Control for dead cells after treatment of cells with20 % (v/v) allyl alcohol for 0.5 h. Regions R1 and R2 show dead and livecells, respectively. Red fluorescence of cells is due to the penetration ofpropidium iodide (PI) into dead cells. Green fluorescence of cells is due tostaining of cell nucleic acids with SYTO9

a

b

c

Fig. 3 a–c Flow cytometry analysis of E. coliDSM 1329 cells incubatedin 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) solution. a Intact cells.b Cells treated with 1 % allyl alcohol for 10min. c Dead cells aftertreatment of cells with 20 % (v/v) allyl alcohol for 30 min. Red fluores-cence is due to reduction of CTC

Ann Microbiol (2015) 65:1007–1016 1011

after incubation with CTC (Fig. 5a,b) and may be consideredas alive but not-respiring (CTC−) cells. This means that twosubpopulations, i.e., actively respiring (CTC+) cells and alivebut not-respiring (CTC−) cells, are present simultaneously inthe population during exponential growth. The average con-tent of ADH-L cells in the exponential phase of batch growthwas about 84 %, while the average content of alive but not-respiring (CTC−) cells was about 18 %. Thus, ADH-L cellswere not sensitive to incubation with 1 % allyl alcohol

solution because of their low respiration rate, as could besupposed.

Additionally, it was shown that the majority (84 %) ofCTC− cells in the exponentially growing population (CTC−

cells in Quadrants 3 and 4 of Fig. 6a) were not dead cells (seeflow cytometry dot plot Quadrant 3 of Fig. 6a). Cells withnon-polarized cell membrane (dead cells) had intensive greenfluorescence of DiBAC4(3) as shown in the flow cytometrydot plot in the Quadrants 2 and 4 of Fig. 6a, b.

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Fig. 4 Dynamics of high alcoholdehydrogenase activity (ADH-H)cells of E. coli DSM 1329(diamonds) and B. megateriumDSM 32 (squares) duringstarvation

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dc

Fig. 5 a–d Subpopulations ofCTC+ cells (region R1) and CTC−

cells (region R2) in batch cultureof E. coliDSM 1329. a 3 h, b 7 h,c 23 h, d control (dead cells). Redfluorescence is due to reduction ofCTC

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The content of live CTC− cells of E. coli was similar to thecontent of ADH-H cells. However, during 7 h of starvation thecontent of CTC− cells of the E. coli population decreasedslightly from 15 ± 2% to 13 ± 3%. Meanwhile, the contentof ADH-H cells in the sample from exponentially growingpopulations of E. coli and B. megaterium decreased to signif-icantly lower values, from 21–28 % to 1–3 % during 2.5 h ofstarvation (Fig. 4). Therefore, ADH-H cells and actively re-spiring CTC+ cells or live but not-respiring CTC− cells belongto physiologically different subpopulations.

Discussion

Analysis of the physiological heterogeneity of natural or in-dustrial microbial populations is an effective way to monitorand control their physiological changes (Ivanov 2010;Lidstrom and Konopka 2010; Rezaeinejad and Ivanov2011). Here, using flow cytometry and specific fluoro-chromes, we showed that there are two physiologically

distinct alive and actively respiring subpopulations in bacterialpopulations of E. coli and B. megaterium growing in mediumwith ethanol as a sole source of carbon and energy. It isimportant to measure the fraction of cells with high ADHactivity during bacterial growth in media containing ethanolbecause knowledge of the physiological heterogeneity of nat-ural and industrial microbial populations is essential to study-ing, modelling and monitoring microbial populations withsubpopulations of distinct biochemical activities. However, itis also important to determine the reason for the simultaneousexistence of subpopulations with high and low ADH activityduring bacterial growth in medium with ethanol. Below, wediscuss three hypotheses and interpretations of the data pre-sented on the different sensitivity of bacterial cells to allylalcohol.

Hypothesis 1: ADH-L cells that persist after 10 minincubation in 1 % allyl alcohol are “survivors” in the dormantstate

ADH-L cells—the fraction of the bacterial population in themediumwith ethanol that persisted after a 10-min incubation in1% allyl alcohol—could be considered at first glance as ”sur-vivors”, i.e., cells in the population that survive upon exposureto stress, starvation, or antibiotics (Booth 2002; Dhar andMcKinney 2007; Lennon and Jones 2011; Patra and Klumpp2013). Persistent forms of bacterial cells accumulate in a pop-ulation by exposure of bacteria to nutrient starvation (Oliver2010), and ADH-L cells accumulated in our experiments dur-ing starvation. However, the “survivors” are usually dormantbacterial cells with slow or almost zero metabolism and theircontent in an exponentially growing population is low (Oliver2010; Lennon and Jones 2011; Balaban et al. 2013; Wood et al.2013), while our data show that the content of ADH-L cells inbacterial populations growing exponentially ranges from 10 %to 85 % and these cells are actively respiring as determined bytheir incubation with CTC. So, bacterial cells growing in themediumwith ethanol as a sole source as carbon and energy andwhich remained alive after 10 min incubation with 1 % allylalcohol are not “survivors”, i.e., dormant or abiotic forms ofcells, but are metabolically active cells that are insensitive to the10-min incubation in 1 % allyl alcohol.

Hypothesis 2: ADH-L cells that persist after 10 minincubation in 1% allyl alcohol are slow-respiring cells

Sub-populations of cells with the different respiration rates areknown to exist (Ryall et al. 2012) because respiration rates candiffer significantly during the bacterial cell cycle (Quiros et al.2007). Differences in the respiration rates of ADH-H andADH-L cells could explain their different sensitivity to incubation in1 % allyl alcohol because a lower respiration rate means lowerproduction of the toxic acrolein. However, our data showed

a

b

Fig. 6 a,b Dot plots of E. coli DSM 1329 cells with red fluorescencerepresenting cells stained with CTC and green fluorescence cells stainedwith DiBAC4(3). a Sample after 10 h of exponential growth in batchculture. b Control, i.e., the same sample with cells killed by incubation ina 20 % solution of allyl alcohol for 30 min

Ann Microbiol (2015) 65:1007–1016 1013

that, during starvation, the contents of sensitive bacterial cellsand actively respiring cells remained virtually unchanged afterincubation in 1 % allyl alcohol. So, the sub-populations ofADH-H and ADH-L cells do not represent populations offast-respiring and slow-respiring cells. Additional proof is thatthere were no ADH-H cells among actively growing and re-spiring cells in the medium with glucose.

Hypothesis 3: ADH-L cells that persist after 10 minincubation in 1 % allyl alcohol do not oxidize external ethanolbut oxidize internal cell carbohydrates

As discussed above, persistingADH-L cells are neither dormant/abiotic forms of cells nor slowly respiring cells, so the mostlogical explanation is that these cells do not oxidize external

ethanol. However, these cells are actively respiring so they mustbe oxidizing other sources of carbon and energy, which can onlybe internal carbohydrate storage. An explanation could also beintracellular storage of polyhydroxyalkanoates in cells but theseusually accumulate as a sink of excessive reducing power andare not metabolized as fast as glucose polymers. Our experi-ments showed that bacterial cells oxidizing glucose are resistantto a 10-min incubation in 1 % allyl alcohol, i.e., ADH activity islow or absent in cells during glucose oxidation. Therefore, thephysiological difference between ADH-H and ADH-L cells canbe explained by supposing that ADH-H cells are oxidizingexternal ethanol but ADH-L cells are oxidizing intracellularcarbohydrates and have low or zero ADH activity (Fig. 7).However, experimental data did not give direct evidence ofintracellular carbohydrates oxidation by ADH-L cells.

ADH-L cell

ADH -H cell

External

ethanol

External

allyl alcohol

Alcohol

dehydrogenase

Storage

carbohydrates

Cell

components

Carbon

dioxide

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ethanol

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allyl alcohol

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acrolein

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Fig. 7 A scheme of thebiochemical differences betweenADH-H and ADH-L cells

1014 Ann Microbiol (2015) 65:1007–1016

Our data showed that ADH activity is low for cells con-suming external glucose. This enzyme is also known not to beinvolved in the aerobic catabolism of bacterial cell storagecompounds such as glycogen or poly-(beta)-hydroxybutyrate.Expression of the adh gene is regulated in E. coli at both thetranscriptional and the translational levels (Holland-Staleyet al. 2000) and the adh gene is subject to carbon cataboliterepression (Arndt and Eikmanns 2007)—known since the1970s–1980s for prokaryotic cells (Lees and Jago 1978). So,oxidation of intracellular storage of carbon and energy canrepress ADH synthesis and initiate deactivation of existingADH enzyme. In our experiments, the external and internalsources of carbon and energy and their catabolic pathwayswere different. An external source of energy was ethanol,inducing ADH activity, but internal sources of energy wereprobably carbohydrates repressing ADH activity. Thishypothesis is close to the ideas of Wang and Levin (2009)on changes in cell metabolism during the bacterial cell cycle.Based on the relatively constant duration of both the C-periodand D-period, and the variable duration of the B-period of theprokaryotic cell cycle, Wang and Levin (2009) proposed that,in the B-period, bacterial cells accumulate intracellularsources of carbon and energy that will be sufficient to supplycarbon and energy during DNA replication and cell division.This feature was considered an adaptation to fluctuations ofnutrients availability in natural ecosystems. Therefore, dis-crimination and enumeration of physiologically alternativesubpopulations of ADH-H and ADH-L cells using flow cy-tometry can be a useful method to monitor the heterogeneityof bacterial populations growing in media with ethanol as asole source of carbon and energy.

Acknowledgments We acknowledge support from Iowa State Univer-sity, and from Nanyang Technological University, Singapore. We aregrateful to Mr. Kumaravel Kandaswamy for his technical assistance inperforming experiments.

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