Impact of Foaming on Surfactin Production by Bacillus Subtilis

8/18/2019 Impact of Foaming on Surfactin Production by Bacillus Subtilis

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126 S. Alonso, P.J. Martin / Biochemical Engineering Journal 110 (2016) 125–133

recirculation loop [3], enabling a robust foaming control whilst

offering an independent in situ foam basedseparation process from

the fermentation parameters.

There is a lack of understanding how cells which have passed

through the foam fractionation process are affected. In particular,

there are few if any reports of whether cell concentration may be

enriched or depleted by the foaming, whether foaming may sepa-

rate cells based on some cell properties, whether cells aredamaged

or howcells behave upon being recycled into the production stage.

The present study aimed therefore to study the impact of foam

recovery systems on the separation and surfactin producing ability

of Bacillus subtilis cells. Firstly, small-scale batch foaming experi-

ments wereemployedwhichenabledfoamingof cellscultured over

a wide range of conditions. The cells were then recycled to assess

the influence of foaming on the subsequent fermentation perfor-

mance. The foamed cultures were varied by age, initial cell density

and initial glucose concentration in order to assess the sensitiv-

ity of cells to foaming over the most common range of conditions.

Secondly, continuous stripping mode foam fractionation experi-

ments wereemployedto investigate the applied process and which

enabled the segregation, and subsequent recycling, of the resulting

cells located in the liquid pool, overflow and foamate compart-

ments (Fig. 1).

2. Material and methods

2.1. Microorganism

B. subtilis BBK006, which was kindly provided by Dr Baker (for-

merly of Oxford Brookes University, UK), was maintained frozen

(in40% [v/v] glycerol at−80 ◦C).This strainwas previously claimed

to be unique compared to other microorganisms in that it only

produced surfactin as unique lipopeptide [18]. The strain was sub-

sequently subcultured on Nutrient Broth (NB, containing 4.3 g/L

meat peptone, 4.3g/L casein peptone, and 6.4g/L NaCl) agar plates,

incubated for 48h at 30◦C, and then preserved at 4 ◦C.

2.2. Culture conditions

A loopful of B. subtilis from a fresh NB agar plate was used

to inoculate a 500 mL Erlenmeyer flask containing 100mL of

M9 medium (Fluka, containing 6.7g/L Na2HPO4, 3 g/L KH2PO4,

3 g/L NH4Cl, 0.5g /L NaCl, 0.12g /L MgSO4, 0.001g /L CaCl2, and

2 mL/L of trace elements). The trace element solution con-

tained the following composition: 52g/L Na2EDTA, 2.2 g/L ZnSO4,

5.44 g/LCaCl2·2H2O,4.9 g/LMnCl2·4H2O,5g/LFeSO4·7H2O,0.35g/L

(NH4)2Fe(SO4)2·6H2O,0.8g/L CuSO4·5H2O,and 1.6 g/L CaCl2.6H2O.

The flask was incubated on an orbital shaker (Innova 42, New

BrunswickSci., NJ,USA) at200 rpmand30 ◦C. Activelygrowing cells

from this culture harvested at different times were subsequently

submitted to foaming processes as reported below.

2.3. Small-scale batch foaming approach

To submit the B. subtilis cells to a foaming process, 45mL of

the culture broth was sparged for 15min under aseptic conditions

in a vessel equipped with a metallic sparger. Sterile air was sup-

plied with an air pump (Interpet, model Aqua AP3, UK) via an in

line 0.2m filter. An air flow rate of 0.5 L/min was controlled via

a rotameter with an air entry at the top of the foaming vessel.

The carryover foam generated in the foaming vessel was collected

via an aseptically attached foam trap fitted with an exhaust vent.

Fig. 2A shows the foaming set-up including the foaming vessel and

foam trap employed for sparging the cells and collecting the foam,

respectively. In many cases, all of the broth was carried over to

the foam trap so systematic segregation of cells into retained and

carried over compartments was not possible. Upon submitting the

cells to the foaming process, all the carryover foam and any liquid

remaining in the foaming vessel were mixed before being har-

vested by centrifugation (11,000 g for 10min). The resultant cell

biomass was reused as inoculum in shake-flask batch cultivations

as reported below.

2.4. Impact of culture age and foaming in culture performance

To study therole of culture ageand foaming in surfactinproduc-

tion, B. subtilis cells harvested at different times (12-, 18-, 24-, and

36-h) were submitted to a foaming process. Upon being submit-

ted to a foaming process (see Section 2.3) and subsequent spinning

down by centrifugation (11,000 g for 10min), cells were employed

as inoculum of shake-flasks. Shake-flask cultivations, inoculated

with 0.019g/L of wet biomass, were conducted in 500mL Erlen-

meyer flasks containing 100mL of M9 mediumsupplemented with

3.5 g/L of glucose. The spent cells submitted to a foaming process

werecomparedwith non-foamingsubmitted spentcells. Thesecul-

tures were subsequently incubatedon an orbital shaker (Innova 42,

New Brunswick Sci.) at 200rpm and 30◦C. Samples were asepti-

callywithdrawnperiodically to determinecellular growth.Biomass

was removed by centrifugation at 11,000 g for 5 min, the cell-freesupernatants being stored frozen (−20 ◦C) until further analysis.

Cultivations were carried out in triplicate as independent trials.

2.5. Impact of initial cell densities and foaming in culture

performance

To study the role of initial cell densities and foaming on biopro-

cess parameters, B. subtilis cells grown for 18h were submitted to a

foaming process (see Section 2.3). After being submitted to a foam-

ing process and subsequent centrifugation (11,000 g for 10min),

cells were employed as inoculum of shake-flasks. Shake-flaskculti-

vations, inoculated with different initial cell biomass values (0.019,

0.038, and 0.076g/L), were conducted in 500 mL Erlenmeyer flaskscontaining 100mL of M9 medium supplemented with 3.5 g/L of

glucose. The spent cells submitted to a foaming process were

compared with non-foaming submitted spent cells. These cultures

were subsequently incubated on an orbital shaker (Innova 42, New

Brunswick Sci.) at 200rpm and 30◦C. Samples were aseptically

withdrawn periodically to determine bacterial growth. Biomass

was removed by centrifugation at 11,000 g for 5 min, the cell-free

supernatants being stored frozen (−20 ◦C) until further analysis.

Cultivations were carried out in triplicate as independent trials.

2.6. Impact of glucose-limited conditions and foaming in culture

performance

To study the role of different glucose concentration along with

foaming,B. subtiliscells grownfor 18h were submittedto a foaming

process (see Section 2.3). To this end,shake-flask cultivations,inoc-

ulated with 0.038 g/L of wet biomass, were conducted in 500 mL

Erlenmeyerflasks containing 100mL ofM9 medium supplemented

with different glucose levels (3.5, 7, 10.5, and21 g/L). The cells sub-

mitted to a foaming process were compared with non-foaming

submitted cells. These cultures were subsequently incubated on

an orbital shaker (Innova 42, New Brunswick Sci.) at 200rpm

and 30 ◦C. Samples were aseptically withdrawn periodically to

determine cell growth. Biomass was removed by centrifugation at

11,000 g for 5 min, the cell-free supernatants being stored frozen

(−20 ◦C) until further analysis. Cultivations were carried out in

duplicate as independent trials.


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S. Alonso, P.J. Martin / Biochemical Engineering Journal 110(2016) 125–133 127

Fig. 1. Experimental workflow carried out in this work. Small-scale batch foaming experiments with further cell recycling were first performed prior to foam fractionation

column experiments with further cell recycling.

Fig. 2. Schematic diagrams of theexperimental small-scale batch foaming (A)and foam fractionation set-ups (B).

2.7. Continuous foam fractionationwith further cell biomass

recycling in batch cultivations

The fermentation broth employed in the foam fractionation

experiments was obtained by culturing two 5-L Erlenmeyer flasks

witha working volumeof 1 L.To thisend, a 0.1 L seedculturegrown

on M9 for 24h (200rpm, 30 ◦C) was employed to inoculate (10%,

v/v) each 5-L Erlenmeyer flask containing 900mL of M9 with a

glucose concentration of 3.5g/L. These cultures were subsequently

incubated on an orbital shaker (Innova 42, New Brunswick Sci.) at

200rpm and 30◦C for 18h .

Continuous stripping mode foam fractionation experiments

were carried out using a bespoke “J-tube” glass foam column

(internal diameter of 51mm, and an exposed height of 310mm)

coupled to a feed and overflow vessels under aseptic conditions. A

schematic diagram of the experimental set-up employed is shown

in Fig.2B. Fermentation broth was pumpedfrom the feeding vessel

to the foam column using a ThermoSci peristaltic pump (Ther-

moSci, model FH 100, UK), at a constant feed rate of 15.35 mL/min.

Overflow of retentate from the bottom pool was controlled via a

peristaltic pump (Watson Marlow, model 503U, UK) set ata rate of

12.8mL/min to attain a constant working liquid level of 90mm in

the foam column. Sterile air was supplied to the column with an


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air pump (Interpet, model Aqua AP3, UK) via a 0.2m filter. Anair

flow rate of 0.1 L/min was controlled via a rotameter with an air

entry at the bottom of the column through a sintered glass disk of

40–60m porosity. Foam was collapsed with a mechanical foam

breaker, being the resulting foamate liquid collected in an asep-

tically clamped 10L glass vessel. Foam fractionation experiments

were carried out under aseptic conditions in duplicate as indepen-

dent trials. With these conditions the bubble residence time in the

column will be around 400 s, the liquid mean residence time in the

pool will be around300s and the liquidmeanresidencetime inthe

foam will be around 100 s.

After performing the foam fractionationfor ∼1.8h, theresulting

liquid was aseptically collected from the different compartments

shown in Fig. 2B: feed,overflow, liquidpooland foamate. Cells from

each compartment were harvested by centrifugation (11,000 g for

10min) and subsequently employed as inoculum of shake-flasks.

Shake-flask cultivations, inoculated with two different initial cell

biomass values (0.03, and 0.06g/L), were conducted in 500mL

Erlenmeyer flasks containing 100mL of M9 medium supplemented

with3.5 g/Lof glucose.These cultures weresubsequentlyincubated

onan orbital shaker(Innova42,New Brunswick Sci.)at 200rpmand

30 ◦C. Samples were aseptically withdrawn periodically to deter-

mine cellular growth. Biomass was removed by centrifugation at

11,000 g for 5 min, the cell-free supernatants being stored frozen(−20 ◦C) until further analysis. Cultivations of cells belonging to

eachfoam fractionation compartment werecarried out in duplicate

as independent trials.

2.8. Cultivation parameters

2.8.1. Specific growth rate

The specific growth rate () (h−1) was calculated according to

Eq. (1) as the slope of the regression line between ln(X.V-X 0.V 0)

versus time (t ) during the exponential growth phase.

(t ) =ln ( X.V ) − ln( X 0.V 0)

(t − t 0) (1)

where X (g/L) and X 0 (g/L) are the biomass concentration at timet (h) and at the beginning of the exponential growth phase t 0 (h),

respectively; V (L) and V 0 (L) are the volumes at time t (h) and at

the beginning of the exponential growth phase t 0 (h), respectively.

2.8.2. Specific volumetric productivity

The specific volumetric productivity (qP ) (mg/gh) was calcu-

lated according to Eq. (2) by dividing the surfactin produced at

a particular time interval by the average biomass (X) during that

specific time interval.

qP mg/g.h

=

msurfactinX.t

(2)

2.8.3. Biomass yield on substrate

The biomass yield on substrate (Y X/S) (g/g) was calculated asthe slope of the regression resulting by plotting the concentration

of biomass formed (g/L) versus the quantity of glucose consumed

(g/L) during the cultivation.

2.8.4. Surfactin yield on substrate

The surfactin yield on substrate (Y P/S) (g/g) was calculated as

the slope of the regression resulting by plotting the concentration

of surfactinproduced (g/L) versus thequantityof glucose consumed

(g/L) during the cultivation.

2.9. Extraction and purification of surfactin

Surfactin was extracted and purified from the fermenta-

tion broth by acid precipitation. After cell removal through

centrifugation(11,000 g for10 min), thesupernatant wassubjected

to an acid precipitation with6 M HCl by adjusting the pH to 2.0 and

kept refrigeratedat 4 ◦C for12 h. Theprecipitant was then collected

by centrifugationat 7600 g for 15min after two washing steps with

distilled water to remove impurities. After drying the precipitant

at 80 ◦C for 24h, the dried surfactin was resuspended in methanol

upon being filtered using a 0.45m filter.

2.10. Analytical methods

Bacterial growth of culture samples was measured spectropho-

tometrically as optical density at 600nm (Shimadzu, UVmini-1240

model) using 0.7% (w/v) NaCl as a blank after centrifugation at

12,000 g for 5 min. Optical density data was converted to cell dry

weight (expressed in grams per litre) using the corresponding pre-

viously obtained calibration curve.

The surfactin content of cell-free culture samples was mea-

sured by high performance liquid chromatography (HPLC) using

an UltiMate3000 UHPLC system (Dionex,California, USA)equipped

with an Acclaim 120-C18 reversed-phase column (Dionex). Analy-

ses were performed using a gradient of solvent A containing 0.1%

trifluoroacetic acid (TFA) (v/v) in water and solvent B consisting

of 0.1% trifluoroacetic acid (TFA) (v/v) in acetonitrile at 30◦

C andat a flow rate of 1 mL/min. The multi-step gradient was performed

according to the following programme: first an isocratic elution at

40% B for 15min, followed by another isocratic elution at 85% B for

20min witha final isocratic elution at 100%B for 5 min. The elution

pattern was monitored at a wavelength of 215nm. The concen-

tration of surfactin was analysed and quantified using the purified

surfactin as previously detailed.

The glucose content of cell-free culture samples was mea-

sured by HPLC using a HPX-87H column (Bio-Rad, California, USA)

coupled to a refractive index detector. 5mM Sulphuric acid was

employed as the mobile phase at a flow rate of 0.4mL/min with

the columntemperature set at 50◦C. Data acquisition and analyses

were performed with Chromeleon v6.8 software (Dionex).

3. Results

3.1. Effect of culture ageing on B. subtilis growth and surfactin

production

Fig. 3 shows how the culture age (cells harvested at early

exponential growth (12 h), mid-exponential growth (18 h), early

stationary growth phase (24h), and late exponential growth

phase (36h)) impacted on the surfactin production outcomes

after a foaming process. Whereas the increase in culture age-

ing was translated into lower specific surfactin production rates

(qs) (∼16mg/g h) but similar surfactin titers (∼0.29g/L) either for

foamed or non-foamed cells (Fig. 3A), biomass yield on glucose

(Y X/S) peaked at ∼0.46g/g by submitting mid-exponential (18 h)and early stationary growth phase cells (24h) to a foaming pro-

cess (Fig. 3B). Submitting the cells to a foaming process therefore

enhanced both surfactin titers (from 0.23 to 0.29g/L with 24h

and 36h cells, respectively) and surfactin specific productivities

(qs) (from 14 to 18mg/gh with 24h and 36h cells, respectively)

even by using aged cells (Fig. 3A). The same positive effect of

foaming in biomass yield on glucose (Y X/S) and surfactin yield on

glucose (Y P/S) can be also observed, suggesting that foaming stim-

ulated the consumption of glucose and its further channelling into

surfactin production (Fig. 3). By using foamed cells harvested at

mid-exponential growth (18h) and early stationary growth phases

(24h ), Y X/S was enhanced up to a value of 0.47 g/g (Fig. 3B). In

terms of Y P/S and qs, cells harvested at mid-exponential growth

phase (18 h) displayedthe highest values eitherfor non-foaming or


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Fig. 3. Impact of culture age on cultivation performance of cells upon being submitted to foaming and non-foaming conditions. (A) Relationship between culture age and

surfactin titer and specific surfactin production rate of cells submitted to foaming and non-foaming conditions. (B)Relationship between culture age and biomass yields on

glucose andsurfactin yieldson glucoseof cells submittedto foaming and non-foaming conditions. Foamedcellsresulting from small-scale batch foaming experiments were

compared with non-foamed cells.

foaming conditions. Comparatively, the volumetric surfactin pro-

ductivity using 18h cells was 0.024g/Lh, which was 33% higher

than productivity achieved using 36h cells (0.018g/Lh). In terms

of production abilities perg cell biomass (qs), harvesting the cells at

late exponentialgrowthphase(36h) displayeda valueof 17mg/gh,

whichwas 32% lower than the qs obtained by using cells harvested

at the exponentialgrowth phase (18 h) (25mg/g h) (Fig.3A). There-

fore, harvesting the cells at mid-exponential growth phase (18 h)

may guarantee a higher stimulation effect of foaming in the sur-

factin production abilities of B. subtilis during a foam recovery

process. A close comparison between RP-HPLC profiles revealed

that foaming greatly enhanced the formation of the different sur-

factin isoforms (Fig. 4). Overall, foaming enhanced not only the

surfactin titer, but also glucose consumption and its further uti-

lization for surfactin production by B. subtilis.

3.2. Culture performance of foamed versus non-foamed cells

under higher initial cell density conditions

As can be seen in Fig. 5A, the final surfactin titer remained atthe same level regardless the double and triple increase in the ini-

tial cell densities. Thus, the qs was barely improved by increasing

the initial cell biomass, resultingin no substantial positive effectby

increasing the initial celldensities. In contrast,foamingdid result in

30% increase in theqs, from levelsaround 18 to 26mg/g.h.Whereas

Y P/S was improved by using low initial cell densities (0.019g/L)

under both foaming and non-foaming conditions, higher Y X/S val-

ues were achieved by using higher initial cell densities (0.076 g/L)

(Fig.5B). Thus, thebiomassformation perunitof glucose consumed

was slightly reduced by increasing the initial cell densities (0.45 vs

0.53 g/g achieved with foamed cell densities of 0.019 and0.076g/L,

Fig. 4. Reversed-phase HPLC chromatograms of surfactin produced by B. subtilis

cells at 12h upon being submitted to a foaming or non-foaming process.

respectively). According to the results, the useof foamedcells ledto

an 1.14-foldtimes enhancement in the finalsurfactin titers attained

whist increasing 1.7- and 1.4-fold times the Y X/S and Y P/S, respec-

tively, in comparison to non-foamed cells. Altogether, it can be

concluded that the joint effect of inoculum size and foaming had

less influence than culture age did.

3.3. Surfactin production by foamed cells vs. non-foamed cells

under glucose-limited conditions

To gain insights into how foamed and non-foamed B. subtilis

cells faced glucose-limited conditions, shake-flask cultures with

different initial glucose concentrations were carried out (Fig. 6).

Addressing the impact of such conditions provided in addition

deeper knowledge on whether increased glucose availability can

increase the final surfactin titer and overall production outputs

upon submitting the cells to a foaming process.

As can be seen in Fig. 6A, glucose concentrations higher than

10.5g/L did not support higher surfactin titers, being 0.37 and

0.41g/L the maximumsurfactintitersachievedafter submitting thecells to a non- and foaming strategy, respectively. However, cells

submitted to a foaming process did lead to higher final surfactin

titers in all the cases considered. Conversely, increased glucose

concentrations did not lead to higher surfactin titers, suggesting

that carbon-limited conditions may improve surfactin production

abilities in B. subtilis cells. Compared to non-foamed cells, cells

submitted to a foaming strategy displayed higher titers in all the

experiments (Fig. 6A). As Fig. 6A shows, a progressive decay in the

qs was observed as the glucose availability was increased. Likewise,

higher qs values were achieved when the cells were submitted to a

foaming process (Fig. 6A).

In terms of yields, major differences in Y X/S w ere found by

increasing the glucose availability. Whereas Y X/S remained stable

at∼

0.21g/g in non-foamed cells regardless the glucose concen-tration, the combination of glucose-limited conditions (3.5g/L)

and foaming stimulated the Y X/S up to 0.28g /g which was pro-

gressively reduced under higher glucose availability (Fig. 6B).

Whereas foamed cells displayed their maximum Y X/S values at

3.5g/L glucose, thehighest Y P/S value was achieved under a glucose

concentration of 10.5g/L by using either foamed or non-foamed

cells. Interestingly, foaming doubled the Y P/S values achieved in

all the cases studied, being for instance 0.062 and 0.099g/g for

10.5g/L with non-foamed and foamed cells, respectively. Alto-

gether, these results suggested that glucose-limited conditions

stimulated surfactin production by channelling more carbon flux

towards surfactin production. The combination of such glucose-

limited strategy with a foaming process enabled to achieve a high

qS while increasing the Y X/S.


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Fig. 5. Effect of initial cell density conditions on cultivation performance of cells upon being submitted to foaming and non-foaming conditions. (A) Relationship between

initial cell density and surfactin titer and specific surfactin production rate of cells submitted to foaming and non-foaming conditions. (B) Relationship between initial cell

density and biomass yields on glucose and surfactin yields on glucose of cells submitted to foaming and non-foaming conditions. Foamed cells resulting from small-scale

batch foaming experiments were compared with non-foamed cells.

Fig. 6. Influence of glucose availability on cultivation performance of cells upon being submittedto foaming andnon-foaming conditions. (A)Relationship between glucose

availabilityand surfactin titerand specific surfactin productionrate of cellssubmitted to foaming and non-foaming conditions. (B) Relationship between glucose availability

and biomass yields on glucose and surfactin yields on glucose of cells submitted to foaming and non-foaming conditions. Foamed cells resulting from small-scale batch

foaming experiments were compared with non-foamed cells.

3.4. Continuous foam fractionation system with further cell

recycling

In order to address the fate of the best surfactin producing cells

within a foam fractionation system as well as to reveal whether

foam recovery systems impact on surfactin production by B. sub-tilis, a foam fractionation experiment with further cell recycling

into batch cultivation was carried out. To this end, the resulting

cells from the foam fractionation compartments shown in Fig. 2B

(feed, retentate overflow, liquid pool, and foamate) were reused

in glucose-limited batch cultivations. Whereas a cell enrichment

of 0.46 was attained in the foamate (0.40g/L of biomass in com-

parison to an initial feed biomass of 0.86g/L), a cell enrichment of

1.15 was attained in the retentate overflow, suggesting that cell

drainage was predominant within the foam fractionation column.

In terms of surfactin recovery, an enrichment of 2.37 (correspond-

ing to a 46% recovery) was achieved in the foamate, whereas a low

surfactin enrichment of 0.78 was found in the overflow.

An overall comparison between the cell growth profiles

obtained by using two different inoculum sizes is shown in Fig. 7.Ascanbeseenin Fig.7A andC, the inductionof both cellular growth

and surfactinproduction occurred earlier in the cultures usingcells

from the foamate than the rest of cultures using cells from other

compartments like overflow and liquid pool. Thus, after a short

adaptation phase (4h), cells submittedto a foaming process started

to grow faster. In fact, cells from the foamate and overflow showed

higher specific growth rates (∼0.5h−1) than those displayed by

non-foamed cells (feed) and cells from the liquid pool (∼0.35 h−1)

(Fig. 7B). The maximum biomass peaked at 14–16 h, representing

the onset of the stationary growth phase and therefore the end of

the surfactin production phase. Non-foamed cells (feed compart-

ment) displayed the poorest production performance (0.22 g/L),

suggesting thatsurfactinproduction inB. subtiliswas stimulated by

using a foam fractionation system (Fig.7B and D). As Fig. 7A shows,

production outputs were not improved despite doubling the ini-

tial cell concentrations (0.06 g/L), resulting in similar final surfactin

titers (e.g., 0.39 and 0.4 g/L under two different cell densities using

cells from the foamate compartment).

In terms of specific growth rates, no meaningful differences

were found between cells from the retentate overflow and cellsfrom the foamate for both inoculum sizes. Cells from such com-

partments displayed a specific growth rate of around 0.5h−1 , in

comparison to a value around 0.35h−1 found for the cells from the

retentate overflow and foamate. There were no major differences

between both inoculum sizes, displaying similar specific growth

rates. Regarding the final surfactin outputs, cells from the foamate

were able to produce higher surfactin titers in comparison to the

cells from the rest of compartments (Fig. 8A). Thus, cells from the

foamate produced 1.81-fold higher final surfactin titers (0.4g/L)

than the cells from the feed compartment (0.22g/L). Interestingly,

these differences were translated into higher qs values, attaining

18mg/gh byusingcells from the foamate with aninitialcell density

of 0.06g/L. (Fig.8C). Likewise, cells from the foamate displayed2.6-

and 1.1-fold higher Y X/S and Y P/S, respectively, than non-foamedcells (Fig. 8D). Overall, whereas cellular growth was not markedly

improved, the surfactin production abilities were clearly triggered

via recycling foamed B. subtilis cells.

4. Discussion

Though second generation of integrated production-foam

recovery systems offer a robust foaming control while an inde-

pendent in situ foam based separation process for biosurfactant

production [3], there is a lack of understanding how spent cell

biomass behaves upon being recycled into the production stage.

To date, most of the ISPR systems have overlooked the contribu-

tion of the spent cells upon being recirculated into the cultivation

stage. Therefore, the foaming strategy developed in the present


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Fig. 7. Cell growth (A) and surfactin production profiles (B) resulting from shake-flask cultivations under glucose-limited conditions with an initial cell density of 0.03 g/L

and using cells from feed, overflow, liquid pool and overflow compartments upon a foam fractionation experiment. Cell growth (C) and surfactin production profiles (D)

resulting from shake-flask cultivations under glucose-limited conditions with an initial cell density of 0.06 g/L and using cells from feed, overflow, liquid pool and overflow

compartments upon a foam fractionation experiment.

Fig. 8. Comparison of final surfactintiters (A), specific growthrate (B), specific surfactin production rates (C), andbiomass yieldson glucose (D)obtainedaftercell recycling

and further shake-flask cultivation of cells from the different foam fractionation compartments: feed, overflow, liquid pool and foamate. Experiments were performed with

initial cell densitiesof 0.03 and 0.06 g/L.

study examined whether submitting cells to a foam recovery sys-

tem may exert a detrimental impact on surfactin production by B.

subtilis.

Results have shown that foaming was effective in improving

the surfactin production abilities displayed by B. subtilis, leading to

2-fold higher surfactin productivity values. Not only cell growth,

but also Y X/S and Y P/S were markedly improved by using foamed

cells. In addition, a clear relationship between enhanced surfactin

production and glucose-limited conditions (< 10 g/L) can be drawn.

Thus, the synergistic combination of foaming and glucose-limited


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Table 1

Comparison of process parameters obtained in thepresent work with integrated schemes for surfactin production.

Microorganism Bioprocessing a pproach (h−1 ) Surfactin titer

(g/L)

Volumetric

productivity

(mg/Lh)

qS (mg/g h) Y X/S (g/g) Reference

B. subtilisATCC21332 B at ch pro duct ion wit h f oam

fractionation

– 2.1 (foam) 10 – 0.13 [2]

B. subtilisATCC 21332 Rotating discs bioreactor – 0.2 3 1 0.19 [7]

B. subtilisDSM 10T Batch production with foam

fractionation

– 3.9 (foam) 18 6 0.27 [14]

B. subtilisDSM 10T Fed-batch production with foam

fractionation

0.31 3.67 (foam) 22 9 0.20 [19]

B. subtilisBBK006 Batch production with foam

fractionation

0.28 2.25 (foam) 10 – 0.26 [11]

B. subtilis

BBK006

Small-scale batch foaming + batch

culture

0.42 0.31 31 26 0.42 This work

Batch culture of non-foamed cells

(Feed)

0.34 0.23 9 8 0.29 This work

Foam fractionation+ batch culture of

foamed cells (Foamate)

0.51 0.41 26 16 0.33 This work

– : Notavailable.

conditions resulted in 1.8-fold higher qs (17mg/gh with 3.5g/L of

glucose) than under higher glucose availability (9.4mg/g h with

21g/L of glucose) (Fig. 6A).

These results clearly suggest that higher surfactin yields andtiters can be obtained under glucose-limited conditions, which is

in accordance withprevious reports [11,19]. In contrast,mostof the

fermentative processes targeting the surfactin production employ

high initial glucose concentrations (20–40 g/L) [2,12], channelling

the glucose overflow toward the formation of side metabolites [20].

As Fig. 6B shows, both Y X/S and Y P/S were progressively reduced

by increasing the glucose availability, revealing a more efficient

biomass formation andsurfactin production per g of carbon source

available under glucose-limited conditions (< 10 g/L).

Continuous foam fractionation experiments were consistent

with results obtained in the small-scale batch foaming approach,

revealing that foamed cells displayed 2-fold higher qs than non-

foamed cells. Comparatively, foamed cells displayed 2.6-fold times

higher specific productivities than the usual values reported in theliterature (Table 1). Furthermore, higher Y X/S valueswere displayed

by foamed rather than non-foamed cells in all the bioprocessing

conditions assessed (Figs. 3B, 5B and 6B), suggesting that a more

effective metabolism with less energy diversion into cell mainte-

nance and biomass formation per g of glucose is achieved under

foaming conditions.

Thus, the present work showed that cells subjected to a foam

recovery system displayed better cell growth and surfactinproduc-

ing abilities, suggesting foaming as a potential approach to boost

the surfactin producing abilities in B. subtilis.

The depletion of cells in the foamate collected from the con-

tinuous stripping mode foam fractionation column was consistent

with that reported by Chen et al. [11] f or the sameB. subtilis strain

and provides insight into the cell interaction withthe foam.Particleseparation by foaming is well established through the technology

of froth flotation [21]. Particles which have hydrophobic surfaces

will tend to attach to the foam air-water interface, whereas par-

ticles with hydrophilic surfaces will not attach to the interface

and remain in the bulk liquid [22]. Once the foamate has been

collectedand collapsedit willbe enrichedin the hydrophobic parti-

cles. Subrahmanyamand Forssberg [23] presentan overviewof the

wide range of possible interactions between particles and draining

foams. Hydrophilic particles will be entrained into the foam inter-

stitial liquid as the same concentration as the bulk liquid pool or

the injected feed, so the foam interface would not be expected to

either enrich or deplete hydrophilic particles in the foamate. The

terminal settling Stokes velocity of a B. subtilis cellis oftheorderof

10−7 m/s so depletion of hydrophilic cells by downwards settling

through the foam in the foam fractionation column would not be

expected. The depletion appears likely to be due to transverse cell

displacement towards the channel centre in the Poiseuille drainage

flow along Plateau borders [24,25]. The effectof this process wouldbe thatthe average celldrainage velocityis greater thanthe average

liquid drainagevelocity andit is likelythat cellsize andmorphology

would affect this.

Reported measurements of the air-water-cell contact angle of

various B. subtilis strains support the notion that the cells are

hydrophilic, and therefore do not attach to the air-water interface.

Ahimou et al. [26] report contact angels below 90◦ for all strains

in all cases, indicating that the cells were hydrophilic. Their results

also showed that the cells may become less hydrophilic when in

a lipopeptide solution (including surfactin) and it was postulated

that this was due to adsorption of the amphiphilic molecules onto

the cell surface whichnegates the cell surface hydrophobicity.Ahi-

mou et al.[27] reported contactangle measurements of a range of B.

subtilis strains and also for cells segregated by physiological statesincluding vegetative cells, spores and segregated by zeta potential.

Allcontactangleswereless than90◦, again indicating the cellswere

hydrophilic. No clear correlations were apparent between contact

angle and physiological state.

In summary, it appears that the cells in this study were most

likely hydrophilic and did not attach to the foam air-water inter-

face. The cells preferentially drained from the liquid entrained in

thefoam dueto particle hydrodynamic effects whichare notlinked

to cellhydrophobicity, butwould be linked to cellsize andmorphol-

ogy.Cell selectionrelatedto physiologicalstate could haveoccurred

due to size or morphology differences. Regardless of the specific

reason, it was shown in this case that subjecting cells to a foaming

process consistently improved their production characteristics and

in particular when the cells were collected in the foamate.From a processing perspective, deciphering which compart-

ment contained the best surfactin producers has enabled to draw

better conclusions about the cell fate within a foam fractionation

column. According to the results, foamed cells from the reten-

tate overflow are well-suited to be recycled into the production

stage, resulting in higher cell growth patterns and surfactin pro-

duction outputs (Fig. 8). It is unknown whether this behaviour is

strain-specific or a particular feature resulting from foaming pro-

cesses. A closer understanding of the phenomenon at molecular

and cell levels may provide insights into the mechanism behind.

Thus, a comparison between the transcriptomic and gene expres-

sion profiles of foamedand non-foamed would help to decipher the

stimulation effect exerted by foaming in B. subtilis cells.


9/9


5. Conclusions

Foaming was found to stimulate cell growth, glucose consump-

tion and surfactin production inB. subtilis. Both surfactin titers and

yields on glucose were enhanced by submitting B. subtilis cells to

a foaming process, revealing for the first time the positive impact

of foam recovery systems on the microbial performance. By com-

bining higher initial cell densities, optimal harvesting time and

glucose-limited conditions, cell growth and surfactin titers were

further improved, achieving a specific surfactin production rate of

26mg/gh under batch conditions. The differential cell growth and

surfactin production abilities found inside the different compart-

ments of a continuous stripping mode foam fractionation column

additionally provided evidence on the potential of foam recovery

systems as bioprocessingapproaches to trigger biosurfactantyields

in microbial cells. Overall, the present study serves as a proof-of-

concept that foam recovery systems and further recycling of the

spent cells can improve the microbial performance, enhancing sur-

factin production abilities in B. subtilis.

Acknowledgements

The authors are grateful for financial support from the UK Engi-

neering and Physical Sciences Research Council (grant EP/I024905)

which enabled this work to be conducted. The authors would also

like to thankthe reviewersfor their constructive andvaluable com-

ments.

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