development of pilot-scale fermentation and stabilisation processes for the production of

http://dx.doi.org/10.1080/09583157.2012.696578

http://www.tandfonline.com

Metarhizium to provide an initial concentration of 5�106 conidia mL�1 in the

culture broth.

Composition of liquid medium

The liquid media used to produce hyphal inoculum and MS of Metarhizium were

composed of a basal salts solution with trace metals and vitamins (Jackson,

McGuire, Lacey and Wraight 1997; Jackson and Jaronski 2009). The defined basalsalts solution used in all liquid cultures contained, per litre of de-ionised water:

KH2PO4, 4.0 g; CaCl2 � 2H2O, 0.8 g; MgSO4 � 7H2O, 0.6 g; FeSO4 � 7H2O, 0.1 g;

CoCl2 � 6H2O, 37 mg; MnSO4 � H2O, 16 mg; ZnSO4 � 7H2O, 14 mg; thiamin,

riboflavin, pantothenate, niacin, pyridoxamine, thiotic acid, 500 mg each; and folic

acid, biotin, vitamin B12, 50 mg each. The pre-culture medium (C:N ratio of 50:1;

[C] �36 g L�1) used for producing the hyphal inoculum of Metarhizium contained

the basal medium supplemented with 80 g L�1 glucose and 9 g L�1 acid hydrolysed

casein (Casamino acids†, Difco Laboratories, Detroit, MI, USA). Glucose solutions(20% w/v) were autoclaved separately from the basal salts solution and added prior

to inoculation.

For 100 L bioreactor studies, the basal medium was supplemented with various

combinations of glucose and acid hydrolysed casein to provide 36 g L�1 carbon and

C:N ratios of 30:1 or 50:1 (Jackson and Jaronski 2009). In addition, 10 mL (0.01%

v/v) of an organic, non-silicone, polypropylene- polyether-based anti-foam (antifoam

204, Sigma Chemical, St Louis, MO, USA) was added prior to autoclaving. Carbon

concentration and C:N ratio calculations were based on 40% carbon in glucose and53% carbon, 8% nitrogen in acid hydrolysed casein Jackson and Jaronski 2009). All

medium components were added and the medium pH adjusted to 5.5 prior to

autoclaving. The pH of the culture medium was uncontrolled during the growth of

Metarhizium cultures.

Fermentation conditions

Hyphal pre-culture inocula of F52 for the 100 L bioreactor studies were obtained by

growing nine replicate 400 mL liquid cultures of Metarhizium in 1 L baffled

Erlenmeyer flasks (#2543-01000, Bellco Glass, Vineland, NJ, USA) using the

previously described pre-culture medium. Pre-cultures were grown for 48 hours at

288C and 300 rpm on a rotary shaker incubator (Innova 4230, New BrunswickScientific, Edison, NJ, USA). These culture conditions produced a thick, homo-

genous hyphal inoculum. Three litres of hyphal inoculum of Metarhizium was used

to inoculate the 100 L bioreactor. For quality assurance, the fermentation broth was

streaked onto nutrient agar plates, incubated for 48 hours at 308C, and visually

evaluated for bacterial contamination.

Microsclerotia of Metarhizium were produced in baffled, 100 L bioreactors

(D100, B. Braun, Allentown, PA, USA). Fermentation temperature was 288C and

three 15 cm diameter Rushton impellors were used for agitation. Fermentationconditions varied during these studies included the agitation rate (150�400 rpm) and

aeration rate (20�60 L min�1 sterile air). Cultures of Metarhizium were harvested

after 4, 5, 6 or 7 days to evaluate the impact of culture age on MS yield, desiccation

tolerance and storage stability. Twenty separate 100 L fermentations of Metarhizium

918 M.A. Jackson and S.T. Jaronski

were conducted during this study and all fermentation conditions compared were

repeated at least two times.

Harvest and drying protocols for MS of Metarhizium

The MS-containing biomass of Metarhizium produced in the 100 L bioreactors was

separated from the fermentation broth using a rotary drum vacuum filter (‘mini-

filter’, Komline-Sanderson, Brampton, ON, Canada; Figure 1). The rotary drum

vacuum filter was pre-coated with 4 kg diatomaceous earth (Hyflo Super Cell†,

Celite Corporation, Lompoc, CA, USA) to produce a 2.5 cm filter bed on the drum

surface. The fungal biomass was separated from the diatomaceous earth filter bed

using a knife moving into the filter bed at a rate of 127 mm per drum revolution and a

drum speed of 1 revolution min�1 (Figure 1). The MS-containing wet filter cakes of

Metarhizium obtained from the rotary vacuum filter were � 70% moisture. The wet

filter cake was granulated using a conical mill (Quadro† Comil† U5, Quadro

Engineering, Waterloo, ON, Canada) with a 6.4 mm screen and a rotor speed of 2000

rpm. This wet granulate of MS-containing biomass was placed on ¼ sheet,

aluminium baking trays to a depth ofB2 mm and air-dried for 16�24 hours

Figure 1. Harvesting and drying process for microsclerotial granules of Metarhizium

brunneum F52. Rotary vacuum filter coated with diatomaceous earth separates microscler-

otia-containing biomass from culture broth. The MS-containing biomass is collected on the

surface of the diatomaceous earth filter bed and then shaved off the filter bed by the advancing

knife. The fungal biomass (�75% moisture) is granulated, spread in a thin layer in an

aluminium tray, and air-dried overnight to B5% moisture.

Biocontrol Science and Technology 919

toB5% moisture. Approximately 65% of the granules produced using this drying

method passed through a 1700 mm opening sieve (12 Mesh) but not a sieve with 600

mm (30 Mesh). Once air-dried toB5% moisture, the MS granules were vacuum-

packaged in polyethylene bags and stored at 48C. Moisture content was determined

using a moisture analyzer (MARK II†, Denver Instruments, Tempe, Arizona) and is

reported as percent of wet weight.

Fermentation, desiccation tolerance and storage stability analyses

Growth and MS production by Metarhizium cultures were evaluated over time by

measuring biomass accumulation and MS formation. Biomass accumulation and

MS concentrations were measured on the day the bioreactor was harvested using dry

weight determination as a measure of biomass and microscopic measurement of MS

concentrations, as previously described (Jackson and Jaronski 2009). Briefly, MS

concentrations were measured by pipetting 100 ml of an appropriate dilution of the

culture broth using a wide-bore pipette and applying the suspension to a glass slide.

The suspension was overlaid with a large cover slip (24�50 mm, #12-545-F, Fisher

Scientific) and the total number of MS in the 100 ml sample was counted

microscopically (Olympus BX60 light microscope). Only well formed MS (50�600

mm diameter) were counted. During sampling, microsclerotial suspensions were

constantly vortexed to ensure sample homogeneity. Replicate samples were taken

from each culture of Metarhizium and all fermentation conditions were replicated at

least twice using the 100 L bioreactors.

The desiccation tolerance and storage stability of air-dried MS granules of

Metarhizium were evaluated by measuring hyphal and sporogenic germination. The

viability and conidia production potential of air-dried MS granules were evaluated

immediately after drying and at 4, 8 and 12 months post-storage. For these analyses,

25 mg of the air-dried MS preparation was sprinkled onto water agar plates and

incubated at 288C. The viability of MS was measured by determining the presence of

hyphal growth after 24 hours incubation. On each water agar plate, 100 MS granules

were observed microscopically with a stereo microscope for hyphal growth and data

was reported as percent germination. These water agar plates were incubated for an

additional 7 days at 288C for analyses of conidia production. To determine conidia

production, water agar plates were rinsed with 10 mL sterile 0.04% polyoxyethylene

sorbitan monooleate (Tween† 80, Sigma) and the plates scraped with a 10 ml plastic

loop (#22-363-600, Fisher Scientific, Pittsburgh, PA, USA) until all material on the

agar surface had been visually dislodged and suspended. The liquid conidial

suspension was pipetted from the rinsed plate and the suspension volume measured.

The conidia concentration of the suspension was determined with a hemacytometer.

Conidia production per gram air-dried MS preparation was calculated and used

for comparison of fermentation and harvesting variables. In addition, conidia

production per gram biomass or per litre fermentation broth of Metarhizium was

calculated for comparison of bioreactor productivity. All air-dried MS granules were

sampled at least twice at each time point tested and each fermentation condition for

MS granule production was replicated at least twice.


Statistical analysis

Mean values were compared for biomass accumulation, MS yields and MS hyphal

and sporogenic germination using ANOVA with Tukey�Kramer mean separation

tests (P50.05). JMP† statistical software (SAS, Cary, NC, USA) was used for

ANOVA analyses.

Results

Fermentation times (4, 5, 6 and 7 days) had no significant effect on biomass

accumulation (19.1�21.1 g L�1) or MS yields (2.6�4.2�107 L�1) by cultures of

Metarhizium grown in 100 L bioreactors using the 30:1 C:N ratio medium. The air-

dried MS granules obtained from cultures harvested at these different fermentation

times all survived drying (100% hyphal germination when rehydrated) and these air-dried MS granules all produced similar concentrations of conidia via sporogenic

germination when compared on the basis of conidia production per litre fermenta-

tion broth (2.3�6.9�1010 conidia L�1). Production of conidia by air-dried MS

granules harvested after 4, 5, 6 or 7 days of fermentation was not significantly

different during the 12-month storage period at 48C when various culture harvest

times were compared at each storage time point or when MS granules from each

culture harvest time were compared during the 12 months of storage (Table 1).

When cultures of F52 were grown in media with a C:N ratio of 30:1 usingdifferent combinations of aeration:agitation, only the lowest aeration:agitation rate

(20 L min�1: 150 rpm) produced significantly lower biomass (Table 2). No significant

difference in MS concentration was observed for any of the other aeration:agitation

conditions tested (data not shown). Immediately after air-drying, conidia production

per litre of fermentation broth or per gram of biomass was significantly higher for

air-dried MS granules from cultures of Metarhizium grown using the lowest agitation

rate (150 rpm) (Table 2). After 4 months of storage at 48C, the air-dried MS granules

from cultures grown using the lowest aeration:agitation protocol produced sig-nificantly higher concentrations of conidia per litre fermentation broth when

Table 1. Stability of air-dried microsclerotial preparations of Metarhizium brunneum F52 from

cultures grown for different fermentation times and stored under vacuum at 48C.

Conidia productiona (�109) (conidia L�1 fermentation

broth)

Storage time

Fermentation Time (Days) Month 0 Month 4 Month 8 Month 12

4 (n�5) 22.6 27.4 67.2 34.8

5 (n�3) 68.7 54.2 74.6 29.1

6 (n�2) 26.8 24.9 74.8 57.9

7 (n�13) 57.7 63.7 40.8 19.7

Notes: Storage stability was measured as conidia production by air-dried microsclerotial preparationsrehydrated and incubated on water agar at 288C.aMean values in columns followed by different lower-case letters and mean values in rows followed bydifferent upper-case letters are significantly different using Tukey�Kramer mean separation tests,P50.05.


compared to MS produced using higher aeration:agitation rates (Figure 2). After

storing MS granules for 4�12 months, there was no significant difference among the

different aeration:agitation protocols tested in terms of conidia production L�1

fermentation broth.

Biomass accumulation in media having a C:N ratio of 30:1 was significantly

greater than in the 50:1 medium (20.4 vs. 11.9 g L�1). The concentrations of MS,

however, were not significantly different in either medium (data not shown). While

air-dried MS granules from the 30:1 and 50:1 C:N ratio media produced similar

numbers of conidia, on a per litre fermentation broth basis, immediately after drying,

the MS granules from media with a 50:1 C:N ratio produced significantly more

conidia per litre of fermentation broth after storage at 48C (Figure 3).When Metarhizium cultures were grown in 100 L bioreactors using media with a

C:N ratio of 30:1, drying protocols had a significant impact on the storage stability

of the MS granules. Microsclerotial granules from cultures supplemented with

diatomaceous earth (DE) prior to dewatering and drying produced fewer conidia per

gram MS granule immediately after drying and after 4 months storage (Table 3).

After 8 months storage, MS granules containing 0�25 g DE per litre fermentation

broth produced similar numbers of conidia that were significantly higher than those

produced by MS granules containing 30�60 g DE per litre fermentation broth. After

12 months of storage, there was no significant difference in conidia production for

any of the MS granules, regardless of DE content (Table 3). These observed trends

were due to a significant loss in conidia production by MS granules without DE over

the 12-months storage period.

When comparing the storage stability of air-dried MS granules having different

moisture levels, conidia production per litre fermentation broth was significantly

higher during the first 4 months of storage for MS granules with higher levels of

moisture (2.6�5%) than MS granules with lower moisture (0�2.5%) levels (Figure 4).

Conversely, after 12 months of storage at 48C, MS granules with lower moisture

content produced significantly more conidia when compared to conidia production

by MS granules with higher moisture levels (Figure 4).

Table 2. Comparison of aeration:agitation rates on culture growth and conidia production by

air-dried microsclerotial granules from cultures of Metarhizium brunneum F52 grown in 100 L

bioreactors using a medium with a C:N ratio of 30:1.

Conidia production (�109)

Aeration (L min�1):

Agitation (rpm)

Biomassa

(g L�1)

Conidia g�1

biomass

Conidia L�1

fermentation broth

20: 150 (n�2) 14.0b 6.1a 85a,b

60: 150 (n�4) 22.0a 4.7a 110a

50: 300 (n�11) 20.8a 1.2b 25b

50: 400 (n�3) 20.2a 1.5b 29b

Notes: Production of conidia by dried microsclerotial granules produced under these culture conditions isused as a relative measure of bioreactor yield.aMean values in columns followed by different letters are significantly different using Tukey�Kramermean separation tests (P50.05).


Discussion

The production of living microbial biocontrol agents using liquid culture fermenta-

tion presents interesting challenges in terms of process development. Measurable

parameters during growth such as biomass accumulation or propagule number serve

to guide media and process development but other factors, such as the desiccation

tolerance and the storage stability of air-dried MS granules, must also be considered.

Microsclerotia of the insecticidal fungus Metarhizium can be used to control soil-

dwelling insects via their production of infective conidia following the incorporation

of MS granules into the soil (Jaronski and Jackson 2008). Our goals in developing a

liquid fermentation process for the production of MS of Metarhizium included

reducing production costs by shortening the fermentation time required to produce a

stable, dry, granular MS product with good shelf-life and the ability to produce

infective conidial inoculum. Two interesting findings from our studies were that (1)

the formation and yield of MS of Metarhizium was not significantly different under

any of the fermentation conditions (C:N ratio, degree of aeration:agitation, duration

of fermentation) tested and (2) all MS granules germinated hyphally after 24 hours

incubation regardless of the fermentation or drying conditions. Even when biomass

yields were significantly lower due to the C:N ratio of the medium or the lower

aeration:agitation rates, MS concentrations in the fermentation broth were

Figure 2. Storage stability of air-dried MS granules from cultures of Metarhizium brunneum

F52 grown using differing aeration:agitation rates. Conidia production by air-dried MS

granules rehydrated and incubated on water agar at 288C for 8 days was used as a measure of

storage stability and is provided on a per litre fermentation broth basis for comparing

bioreactor productivity.


Figure 3. Comparison of storage stability for air-dried MS preparations from cultures of

Metarhizium brunneum F52 grown in media with carbon-to-nitrogen (C:N) ratios of 30:1 or

50:1. Cultures were grown for 5 days in 100 L bioreactors using a Casamino acid-

supplemented basal salts medium. Conidia production L�1 fermentation broth was used as

a measure of bioreactor productivity.

Table 3. Impact of diatomaceous earth content on conidia production by air-dried MS

granules of Metarhizium brunneum F52 following rehydration and incubation on water agar at

288C.

Conidia production (�108) (conidia g�1

air-dried microsclerotial granules)

Storage time

Diatomaceous earth (g/L fermentation broth) Month 0 Month 4 Month 8 Month 12

0 (n�2) 42.5a,A,B 71.5a,A 31.0a,B,C 6.0C

25 (n�2) 19.0b 30.2b 18.0a,b 6.0

30 (n�5) 6.9b 5.8b 7.2b 4.9

50 (n�7) 7.4b 5.7b 7.4b 6.8

60 (n�3) 5.0b 3.6b 14.3b 4.6

Notes: Production of conidia g�1 air-dried MS granule is used for comparison.Mean values in columns followed by different lower-case letters and mean values in rows followed bydifferent upper-case letters are significantly different using Tukey�Kramer mean separations test,P50.05.


unaffected. The lack of significant differences in MS yields for the culture conditions

tested focused our attention on the storage stability and conidia production potential

of the MS granules produced during this study.

Fermentation time is a critical growth parameter in terms of MS formation.

Previous liquid culture studies on MS formation by plant pathogenic fungi showed

that a differentiation process takes place following balanced growth where diffuse

fungal hyphae aggregate to form sclerotia with increased MS melanisation and

compaction as cultures aged (Jackson and Schisler 1995; Shearer and Jackson 2006;

Jackson and Jaronski 2009). The importance of MS compaction and melanisation in

regard to storage stability and conidia production potential is unknown. Under the

conditions of these studies, MS formation and development by Metarhizium cultures

were similar to plant pathogenic fungi, with MS formation initiated after 2 days

growth, and compaction and melanisation subsequently increasing with a longer

fermentation time (data not shown). Harvesting and drying MS-containing biomass

after fermentation times of 4�7 days had no significant impact on the shelf-life or

conidia production potential of the MS granules (Table 1). These results suggested

that 4-days fermentations are adequate for the production of stable MS by cultures

of Metarhizium. While the importance of MS melanisation or compaction was not

significant in our storage and conidia production assays, these processes may be

important under field conditions. Previous studies on fungal melanins have shown

Figure 4. Storage stability of air-dried microsclerotial preparations of Metarhizium brunneum

F52 from cultures dried to various moisture levels and stored under vacuum at 48C. Storage

stability was measured as conidia production by air-dried microsclerotial preparations

rehydrated and incubated on water agar at 288C.


that these compounds can mitigate the deleterious effects of UV radiation,

temperature and moisture fluctuations, free radicals and metal ions (Butler and

Day 1998). In addition, melanised fungal cells were shown to be more resistant to

microbial attack in the soil, and melanins and their shunt products have been shown

to possess antimicrobial properties against antagonist organisms (Bloomfield and

Alexander 1967; Bell and Wheeler 1986). Bioassays comparing young, non-

melanised MS and older, melanised MS granules in live soil are warranted todetermine the effect of fermentation time and subsequent MS melanisation and

compaction on persistence in soil and insect biocontrol efficacy.

Aeration and agitation have potential to impact culture growth and MS

formation by cultures of Metarhizium. The shearing forces generated by impellors

in stirred tank bioreactors have been shown to impact the growth patterns of

filamentous fungi (Gibbs, Seviour and Schmid 2000; Amanullah 2002). Aeration

rates can also influence the growth characteristics and morphology of filamentous

fungi by limiting oxygen availability or altering metabolic pathways (Cui, van der

Lans and Luyben 1998; Papagianni 2004). Our studies showed that a low

aeration:agitation rate reduced biomass accumulation by cultures of Metarhizium

but a low agitation rate (with high or low aeration rates) improved the production of

conidia production by air-dried MS granules immediately after drying and during the

first 4 months of storage (Figure 2). These results suggested that a slower impellor

speed may have a beneficial effect on the subsequent ability of MS to produce

conidia. Future studies are planned to evaluate the interactions between agitatorspeed and aeration rate in affecting dissolved oxygen and carbon dioxide levels and

their impact on the progression of MS formation and maturation.

Earlier shake-flask studies with cultures of Metarhizium showed that media with

C:N ratios of 30:1 and 50:1 both produced high concentrations of MS (Jackson and

Jaronski 2009). Our pilot-scale, 100 L bioreactor studies demonstrated that while

biomass accumulation was significantly higher in the 30:1 C:N ratio medium, the

concentration of MS produced in 30:1 or 50:1 media was similar and the conidia

production potential was higher for MS granules from cultures of Metarhizium

grown in media with a C:N ratio of 50:1 (Figure 3). Future medium optimisation

studies will focus on intermediate nitrogen levels (media with C:N ratios between

30:1 and 50:1) that could potentially increase biomass production without

diminishing storage stability or conidia production by MS granules of

Metarhizium. The biocontrol efficacy of the MS granules produced under these

varied nutritional conditions will also be evaluated.

Drying protocols can have a significant impact on the desiccation tolerance andstorage stability of fungal propagules (Connick et al. 1996; Zhang et al. 2006;

Jackson and Payne 2007; Jin and Curtis 2011). For entomopathogenic fungi that

actively infect their insect host, it is imperative that the infectious fungal propagule

remain viable and capable of completing the infection process (Jaronski 1997;

Wraight, Jackson and DeKock 2001). Fungal MS are overwintering structures well

suited for surviving adverse environmental conditions such as desiccation. The use of

these MS-based biocontrol agents requires that they not only remain viable but also

capable of sporulation to produce infective conidia. Obviously, as the proportion of

MS granules producing abundant conidia on their surfaces decreases, the probability

that an insect crawling through a treated zone acquires an infectious dose of conidia

when it encounters a granule also decreases.


The separation of fungal biomass from culture broth for drying and use as a

biocontrol agent generally requires the addition of an inert component that improves

filtration and that maintains separation between the fungal propagules during the

drying process. If propagules such as fungal spores or MS are not separated during

drying, cells physically adhere to each other reducing the effective number of

infective propagules. Granulation of adhering cells can also lead to propagule

mortality as cell walls and membranes are ruptured. Diatomaceous earth is an

excellent, low-cost, non-compressible, inert, filter aid that has been used successfully

to separate fungal spores and microsclerotia (Jackson and Schisler 1995; Jackson

et al. 1997). While conidia production per gram air-dried MS granule was initially

higher with no diatomaceous earth added, all MS granules produced similar

numbers of conidia g�1 dry preparation after 12 months of storage. This was due

to a significant loss in conidia production by dried MS granules without

diatomaceous earth (Table 3). The MS granules containing 30�60 g diatomaceous

earth per litre produced similar conidia concentrations throughout the 12-months

study, suggesting that conidia production trends of MS granules may be non-linear.

After 12 months of storage, even cultures supplied with 60 g L�1 diatomaceous earth

(�75% less MS biomass per gram dry formulation) produced numbers of conidia

comparable to dried MS formulations with no diatomaceous earth. Further studies

are needed to determine the minimal concentration of MS biomass required per

gram dry formulation to produce stable concentrations of conidia of Metarhizium.

Determination of the minimal number of conidia per MS granule needed to cause

a fatal infection in the target insect would be a useful guide for developing

formulations that maximise the use of MS granules. Certainly, application rates will

be dependent on the insect pest and its habit in the soil. In one set of bioassays using

larval sugarbeet root maggot in a non-sterile clay soil, MS granules of Metarhizium

incorporated at the rate of 0.2 mg g�1 soil, resulted in 57 and 100% mortality after 1

and 2 weeks of exposure (Jaronski and Jackson 2009). That application rate yielded a

MS granule concentration of 1 granule 6 cc�1 soil with each MS granule (0.6�1.2

mm diameter) producing an estimated 1.1�106 conidia. When conidial powders of

Metarhizium were thoroughly incorporated into the same soil, similar levels of insect

mortality were not reached until a conidia concentration of 5�8�106 conidia cc�1

soil were applied (Jaronski 2007). These data provide an application baseline for this

Metarhizium strain against larvae of the sugarbeet root maggot.

The moisture content of fungal biocontrol agents has been shown to significantly

impact the shelf-life of these products (Hedgecock, Moore, Higgins and Prior 1995;

Jin and Curtis 2011). In this study, all MS granules were dried to B5% moisture. MS

granular formulations containing more than 2.5% moisture produced more conidia

per litre of fermentation broth immediately after drying, but the drier MS granules

(B2.5% moisture) produced significantly higher conidia concentrations after 4

months of storage despite in vacuo conditions (Figure 4). The improved storage

stability of MS granules with lower moisture content suggests this is a critical

parameter that must be controlled during drying and packaging. While these storage

studies were conducted at 48C, MS granule storage at higher temperatures will likely

benefit from lower moisture content as this will deter microbial growth and product

contamination by reducing basal metabolic activity in MS granules.


Conclusion

Our studies have shown that MS of Metarhizium can be mass produced using stirred-

tank bioreactors without reducing product quality or stability. While our studies have

provided general parameters critical to process scale-up, improvements can likely be

made in optimising oxygen requirements, agitation rates and medium composition

for improved yields and reduced fermentation times. The optimisation of drying and

formulation processes also shows promise for improving the storage stability and

extending the efficacy of air-dried MS granules of Metarhizium. The development of

fermentation processes that reduce production and formulation costs coupled with

better product stability and maximisation of MS granule efficacy will enhance the

potential for these MS granules of Metarhizium to become a commercially-available

product for insect control.

Acknowledgements

The authors express their sincerest appreciation to Angela R. Payne for her outstandingtechnical assistance on this project.

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