fungal isolate 
 bvk - sma-2 was obtained from a soil sample collected 
 in buffalo valley , oklahoma .
a blast analysis of its its sequence 
 data demonstrated that the isolate was 99% identical to b. 
 ochroleuca .
for the purpose of comparing its growth and metabolite 
 profiles using two solid - phase culturing techniques , the b. 
 ochroleuca was inoculated onto cheerios breakfast cereal 
 in ( a ) 50  1 l erlenmeyer flasks and ( b ) 3 mycobags ( each 53 
 cm  35 cm ) .
the amounts of cheerios and inoculum introduced 
 into the mycobags and flasks were scaled relative to one another in 
 order to ensure the ratios of inoculum to culturable surface area 
 in the two types of vessels were approximately equivalent .
both sets 
 of cultures were incubated at 25 c under identical lighting 
 for 4 weeks .
the secondary metabolites from both sets of b. 
 ochroleuca cultures were extracted with etoac for chemical 
 analysis and biological testing .
extracts were dissolved in 
 meoh  h2o ( 9:1 ) , and 
 the supernatants were subject to lc - pda - esims analysis . the resulting 
 metabolite profiles for both sets of b. ochroleuca cultures were judged to be similar ( figure 1 ) . as an extension of this observation , the secondary metabolite 
 profiles for
several other fungi grown under both types of solid - phase 
 culture conditions were also compared ( figure 
 s1 ) . in the majority of cases ,
the fungi exhibited remarkably 
 similar metabolite profiles , indicating that mycobags are a suitable 
 alternative to flask - based fermentation .
significantly , the culturable 
 surface area of one mycobag ( 1855 cm ) is equivalent to 
 that of approximately 18  1 l erlenmeyer flasks . considering 
 that mycobags are relatively inexpensive ( 0.500.60 
 usd / bag with one hydrophobic gas exchange patch ) , occupy limited vertical 
 space ( mycobags can be incubated in a relatively flattened position ) , 
 and require no cleaning ( mycobags are disposed of after autoclaving ) , 
 their use for solid - phase fungal cultures presents many advantages .
effects 
 of different culture vessels on the secondary metabolome 
 of b. ochroleuca grown on a cheerios - based medium . 

the b. ochroleuca cultures were grown in 1 l erlenmeyer 
 flasks ( a ) or mycobags ( b ) .
the resulting secondary metabolome profiles 
 ( pda detection at 200400 nm ) from the flask - derived ( c ) and 
 mycobag - derived ( d ) cultures reveal relatively modest changes in their 
 respective metabolite composition .
after comparing the b. ochroleuca secondary 
 metabolomes 
 generated under both sets of conditions , the extracts were pooled , 
 and the bioactive compounds were purified using normal - phase , size - exclusion , 
 and reversed - phase chromatography methods to yield four known o - linked glycosylated polyketides , tmc-151s c  f ( 14 ) , and three new metabolites , named 
 bionectriols b  d ( 57 ) .
metabolites 14 were identified by comparisons of 
 their ms data , h and c nmr spectra , and optical 
 rotation values to published values .
compound 5 was obtained as a colorless , amorphous 
 solid that yielded an adduct ion with m / z 625.3934 [ m + na ] under hresims conditions , which was 
 determined to represent the molecular formula c32h58o10na ( calcd 625.3928 ,  0.96 ppm ) .
the h , c , and hsqc nmr data ( table 1 ) suggested the presence of nine methyl , three methylene , 
 six methine , one oxygenated methylene , eight oxygenated methine , and 
 four olefinic carbons .
the hmbc spectrum displayed a correlation from 
 h3 - 19 to c-1 , indicating the presence of an ester carbonyl 
 carbon that had not been detected in the c spectrum . 

the other features of the c spectrum of 5 were similar to those for 1 , except that the sugar 
 alcohol moiety and one propenyl subunit ( equivalent to c-2 , c-3 , and 
 c-21 in 1 ) were missing ( figure s5 ) .
the carbon chemical shifts at c 62.9 , 68.5 , 
 72.7 , 75.7 , 78.3 , and 102.6 denoted the presence of a hexopyranose . 

an hmbc correlation from h-1 to c-11 indicated that the hexopyranose 
 moiety was attached to c-11 .
additional hmbc correlations that were 
 critical for verifying the planar structure of 5 are 
 illustrated in figure 2 .
on the basis of 2d 
 roesy correlations between h3 - 20 and h-6 , as well as h3 - 22 and h-10 ( figure 2 ) , both the c-4c-5 
 and c-8c-9 olefins were determined to be e - configured .
assignments based on h , c , and hsqc nmr ( c 100/h 
 400 mhz ) experiments at room temperature .
the relative configuration of the glycan moiety was 
 ascertained 
 based on gated decoupling and roesy nmr experiments . in the gated 
 decoupling experiment ( figure s6 ) , the 
 coupling constant between the anomeric carbon and hydrogen atoms was 
 determined to be jc-1,h-1 = 154.5 ( < 160 ) , indicating a -orientation .
roesy correlations between h-1 and h-2 , 
 h-1 and h-3 , h-1 and h-5 , h-2 
 and h-3 , h-2 and h-5 , and h-3 and 
 h-5 were observed ( figure 2 ) , which 
 were consistent with a mannose moiety .
additional comparisons between 
 our carbon chemical shift data ( c-1 to c-6 ) and those 
 published for similar compounds further 
 corroborated this assignment .
further efforts to address the absolute 
 configuration of the presumably polyketide portion of the metabolite 
 proved unsuccessful ; thus the absolute configuration of the aglycan 
 component of 5 was not determined .
the hresims data for 6 provided an adduct ion 
 [ m + na ] at m / z 929.5474 , 
 which represented the molecular formula c46h82o17na ( calcd 929.5450 ,  2.58 ppm ) .
the h , c , and hsqc nmr spectra ( table 1 ) suggested the presence of 12 methyl , three methylene , seven methine , 
 three oxygenated methylene , 13 oxygenated methine , six olefinic , and 
 two ester carbonyl carbons .
the c nmr spectrum of 6 was similar to that of 3 with the major exceptions 
 being the presence of additional methyl ( c 14.7 ) , 
 methine ( c 44.7 ) , and oxygenated methine ( c 82.1 ) groups ( figures s13 and s14 ) .
hmbc correlation data were employed to deduce the planar structure 
 of 6 , which revealed that the new carbons were located 
 adjacent to the c-1 ester .
a roesy experiment was performed revealing 
 correlations between h3 - 24 and h-6 , h3 - 26 and 
 h-10 , and h3 - 28 and h-14 , thus verifying that the olefins 
 were e - configured .
in addition , correlations between 
 h-1 and h-2 , h-1 and h-3 , h-1 
 and h-5 , h-2 and h-3 , h-2 and h-5 , 
 and h-3 and h-5 were detected , indicating that the 
 glycan was a mannose .
a coupling constant of jc-1,h-1 = 154.1 was observed 
 for the anomeric c  h bond supporting a -orientation .
further efforts to substantiate the absolute configurations of 
 mannose and mannitol in 6 were pursued using a modification 
 of kohno s approach .
compound 6 was hydrolyzed in 0.4 m naoh , the reaction mixture was neutralized , 
 and products were partitioned against etoac to yield 7 .
the water layer was dried under vacuum and reacted with ac2o in anhydrous pyridine , and the products were extracted with 
 etoac to yield 8 , hexa - o - acetylmannitol 
 ( figures s29 , s30 ) .
a comparison of the 
 optical rotation values for 8 ( [ ]d25 + 24 [ c 0.13 , chcl3 ] ) and authentic hexa - o - acetyl - d - mannitol ( [ ]d25 + 26 [ c 0.693 , chcl3 ] ) suggested 8 was d - configured .
another new metabolite was purified from the fungal crude extract ; 
 however , its hresims , 1d and 2d nmr ( figures s2127 ) , and optical rotation data were identical to those of the base - hydrolysis 
 product 7 .
compound 7 was subjected to acid 
 hydrolysis in 1 m methanolic hcl at 70 c and extracted with 
 etoac .
the water layer was neutralized and dried under vacuum to yield 9 , which was determined to be methyl--mannopyranoside 
 based upon comparisons of its c nmr spectrum (  
 102.7 , 72.0 , 72.5 , 68.4 , 74.4 , 62.7 , and 55.2 , figure s32 ) with published data . a comparison of the optical rotation value for 9 ( [ ]d25 + 60 [ c 0.09 , h2o ] ) with authentic methyl--d - mannopyranoside ( [ ]d25 + 80 [ c 10 , h2o ] ) suggested that 9 is d - configured .
during 
 the course of these studies , colorless , needle - like crystals of 7 were fortuitously obtained from a concentrated methanol 
 solution of the metabolite .
this provided an opportunity to use x - ray 
 diffraction analysis to verify the planar structure of 7 , as well as assign its absolute configuration ( figure s31 ) .
compounds 3 , 4 , and 6 produced 
 modest inhibition of c. albicans cell growth with 
 ic50 values of 36.3  11.3 , 41.0  5.6 , and 24.1 
  4.5 m ( 30.8  9.6 , 33.6  4.6 , and 21.8  
 4.1 g / ml ) , respectively .
the biofilm - inhibiting positive control 
 etya ( 5,8,11,14-eicosatetraynoic acid ) and all of the other purified 
 metabolites were inactive in this assay at concentrations of up to 
 100 m .
however , all the compounds exhibited biofilm inhibition 
 properties with ic50 values ranging from 1.1 to 5.0 m 
 ( 0.9 to 3.0 g / ml ) , and most of them displayed increased inhibition 
 relative to etya ( table 2 , figure 4 ) . the potencies of the b. ochroleuca - derived biofilm inhibitors are similar to 
 waikialoid a ( ic50 value of 1.4 m ) , yet are 2 to 5 times more potent than the mutanobactins 
 and shearinines .
all of the compounds were tested 
 for their abilities to synergistically enhance the activity of amphotericin 
 b against c. albicans .
fici ( fractional inhibitory 
 concentration index ) values for all seven compounds were found to 
 be < 0.5 , indicating synergistic effects with amphotericin b. all 
 of the compounds were found to enhance the potency of amphotericin 
 b up to 4-fold ( table 3 ) .
effects of compounds 6 and 7 ( 2.5 m ) 
 on c. albicans sc5314 biofilm formation .
the c. albicans cultures were prepared in rmpi 1640-mops medium 
 and treated with compounds 6 ( a ) , 7 ( b ) , 
 or dmso vehicle ( c ) before incubating at 37 c for 48 h. panels 
 a  c illustrate representative fluorescent images taken from 
 triplicate cultures .
ic50 values are expressed 
 as the concentrations corresponding to 50% of the maximum biofilm 
 formation .
the mic was defined as the lowest 
 concentration causing prominent growth reduction ( 80% reduction 
 in the metabolic activity ) .
fici = fica + ficb = ( mica  combination / mica  single ) + ( micb  combination / micb  single ) .
nmr spectra were obtained on a varian unity inova spectrometer ( 400 
 mhz for h and 100 mhz for c ) with a broad 
 band probe at 20  0.5 c using methanol - d4 ( cambridge isotope laboratories , inc . ) as the solvent . 

hresims spectra were collected on an agilent 6538 ultra high definition 
 accurate - mass q - tof system .
the lc - ms data were acquired on a shimadzu 
 uflc coupled to a quadrupole mass spectrometer using a phenomenex 
 kinetex c18 column ( 3.0 mm  75 mm , 2.6 m particle 
 size ) and mecn  h2o ( with 0.1% hcooh ) gradient ( 1:9 
 to 0:10 in 12 min ) followed by a 100% mecn wash .
column chromatography 
 was conducted using silica gel 60 ( 4063 m particle 
 size ) and sephadex lh-20 .
tlc silica gel 60 f254 plates 
 from emd chemicals inc . were used for tlc .
separations were also carried 
 out using a shimadzu hplc system equipped with lc-6ad solvent delivery 
 pumps coupled to an spd-10av uv  vis detector or a waters system 
 equipped with a 1525 binary hplc pump coupled to a 2998 pdad detector . 

both systems utilized phenomenex c18 columns ( 21.2  
 250 mm or 10  250 mm , 5 m particle size ) for preparative 
 runs .
x - ray diffraction data for compound 7 were collected 
 using a diffractometer with a bruker apex ccd area detector and graphite - monochromated 
 mo k radiation (  = 0.710  73  ) .
a 
 soil sample was collected from beneath a granite monument on the grounds 
 of one of the author s ( j.b.k . )
the sample was processed using the method described 
 by du et al . , and
the large - ribosomal - subunit internal transcribed spacer 1 ( its1 ) region 
 of the rdna gene was sequenced at the laboratory for genomics and 
 bioinformatics , ou health sciences center .
sequence data for the isolate 
 ( genbank accession km000126 ) was compared by blast analysis to sequences publicly 
 available through the ncbi database . for the 1 l erlenmeyer 
 flask cultures , an 80 ml volume of cheerios breakfast cereal
was added 
 to each flask , and the flasks were autoclaved using a solid - cycle 
 at 121 c for 30 min .
after the flasks were cooled to room temperature , 
 a 48 ml aliquot of autoclaved 0.3% sucrose with 0.005% chloramphenicol 
 was pipetted into the flasks to rehydrate the cereal .
one 5 
 mm  5 mm chunk of the fungal isolate grown on an agar plate 
 was added to each flask , and the cultures were incubated for 4 weeks .
when mycobags were used as the fermentation vessels , cheerios breakfast 
 cereal was preautoclaved using a solid - cycle at 121 c for 30 
 min . after cooling ,
a 500 ml volume of cheerios was added to each 
 bag , the openings of the bags were folded over once , the folds were 
 sealed with clamps , and bags were autoclaved using a liquid - cycle 
 at 121 c for 30 min .
eighteen 5 mm  5 mm chunks 
 of fungal culture grown on an agar plate were prepared and mixed with 
 the cereal . the cereal was then rehydrated with 850 ml of an autoclaved 
 solution consisting of 0.3% sucrose with 0.005% chloramphenicol .
the 
 bags were placed flat to facilitate the formation of a monolayer of 
 cheerios across the bottom of the bag , and the cultures were incubated 
 for 4 weeks .
the cultures were 
 homogenized and extracted with etoac . the etoac extract ( 53.8 g )
was 
 subjected to silica gel flash chromatography with elution carried 
 out using a step gradient of hexane  etoac ( 100:0 , 50:50 , 0:100 ) 
 and etoac  meoh ( 50:50 , 0:100 ) to yield five fractions .
bioassay 
 analysis of the resulting fractions revealed all of the biofilm inhibition 
 activity was retained in fraction 4 ( fr.4 ) .
accordingly , fr.4 ( etoac  meoh , 
 50:50 , 4.6 g ) was subjected to sephadex lh-20 column chromatography 
 and eluted with meoh  dcm ( 1:1 ) , providing 16 subfractions . 

recombination of the 16 fractions based on their tlc profiles yielded 
 four subfractions ( fr.4 - 14 - 4 ) .
fr.4 - 2 ( 0.9 g ) was the only 
 fraction exhibiting biofilm inhibition .
further purification of its 
 components using c18 hplc ( 250 mm  20.2 mm , 5 m ) 
 with a meoh 
h2o gradient ( from 80:20 to 100:0 ) , 
 as well as isocratic c18 hplc ( 250 mm  10 mm , 5 m ) 
 with mecn  h2o containing 0.1% hcooh ( 55:45 , 72:28 , 
 or 78:22 ) , yielded four known [ tmc-151 c  f ( 14 ) ] and three new polyketide glycosides [ 5 ( 3 
 mg ) , 6 ( 12 mg ) , and 7 ( 4 mg ) ] .
a 1 ml aliquot of 
 0.4 m naoh was added to 6 ( 10 mg ) , and the mixture was 
 stirred at room temperature for 2 h. after the reaction mixture was 
 neutralized with 1 m hcl , 1 ml of water was added and the mixture 
 was partitioned with etoac ( 5  3 ml ) .
the pooled etoac layers 
 were reduced under vacuum to yield 7 ( 7.4 mg ) .
the water 
 layer was dried under vacuum and acetylated as described in the following 
 section .
the solid product from the 
 water layer was dissolved in 800 l of anhydrous pyridine with 
 200 l of acetic anhydride .
the mixture was stirred at room 
 temperature for 2 h. when the reaction was completed , 5 ml of etoac 
 was added and the solution was washed repeatedly with h2o ( 5  2 ml ) .
a solution of 1 
 m methanolic hcl ( 800 l ) was added to 7 ( 3.7 mg ) , 
 and the solution was refluxed at 70 c for 1.5 h. after the reaction 
 mixture was neutralized with 1 m naoh , 1 ml of water was added , and 
 the solution was partitioned against etoac ( 5  3 ml ) .
the water 
 layer was evaporated under vacuum to provide 9 ( 1.0 
 mg ) .
a colorless prism - shaped crystal of dimensions 0.420  0.230 
  0.100 mm was selected for structural analysis .
the sample was 
 cooled to 100 k. cell parameters were determined from a nonlinear 
 least - squares fit of 2682 peaks in the range 2.26 <
 
 < 17.05. a total of 20  189 data were measured in the 
 range 1.129 <  < 19.780 using  oscillation 
 frames .
the data were corrected for absorption by the empirical method , 
 giving minimum and maximum transmission factors of 0.963 and 0.991 . 

the data were merged to form a set of 4239 independent data with r(int ) = 0.1301 and a coverage of 50.9% .
the monoclinic 
 space group c2 was determined by systematic absences 
 and statistical tests and verified by subsequent refinement .
the structure 
 was solved by direct methods and refined by full - matrix least - squares 
 methods on f. positions of hydrogens bonded to carbons were initially determined 
 by geometry and refined by a riding model .
hydrogens bonded to oxygen 
 atoms were located on difference maps and were refined with a riding 
 model .
hydrogen atom displacement parameters were set to 1.2 
 ( 1.5 for methyl ) times the displacement parameters of the bonded atoms . 

a total of 489 parameters were refined against 210 restraints and 
 4239 data to give wr(f ) = 0.4094 and s = 1.142 for weights of w = 1/[(f ) + ( 0.1400p ) + 80.00p ] , where p = [ fo + 2fc]/3 .
the final r(f ) was 0.1647 for the 3058 observed [ f > 4(f ) ] data .
the final difference map 
 had maxima and minima of 0.889 and 0.427 e /  , respectively .
the intensity data were 
 truncated to 1.05  resolution because data in higher resolution 
 shells all had i/(i) < 2.0 .
atoms o10 and c6 were disordered 
 ( the occupancies for these atoms refined to 0.53 and 0.47 for the two structures 
 illustrated in figure s31 ) .
restraints 
 on the positional parameters of the disordered atoms and the displacement 
 parameters of all atoms were required .
the x - ray crystallographic data of 8 have been deposited in the cambridge crystallographic data center 
 under accession number ccdc 1007026 .
the data can be accessed free of 
 charge at http://www.ccdc.cam.ac.uk/. the c. albicans strain sc5314 was cultured in brain heart infusion medium ( bhi medium , 
 becton dickinson ) or rpmi-1640 plus mops medium [ rpmi-1640 medium 
 ( sigma ) buffered to ph 7.0 with 0.17 m mops 3-(n - morpholino)propanesulfonic 
 acid ( sigma ) ] as required .
the effects of compounds on the growth 
 of c. albicans were tested using the method described 
 in the nccls 2008 clsi m27-a3 guidelines .
cells of c. 
 albicans sc5314 were cultured in bhi medium ( becton dickinson ) 
 at 37 c overnight .
the cells were pelleted by centrifugation , 
 washed with sterile pbs ( phosphate - buffered saline , ph 7.4 ) , and resuspended 
 in rpmi-1640 plus mops medium .
test compounds were prepared in dmso 
 at stock concentrations of 20 mm before being serially diluted in 
 rpmi-1640 plus mops medium for testing .
aliquots of yeast suspension 
 ( 100 l containing 2.5  10 cells ml ) were added to the medium containing the diluted compounds or dmso 
 [ final concentrations did not exceed 1% ( v / v ) ] before being transferred 
 to 96-well plates ( corning ) .
after 48 h of incubation at 37 c , 
 the viability of the yeast was measured using the xtt assay . in brief , 
 yeast cells were treated with 0.1 mg ml xtt at 
 37 c for 1 h. absorbance measurements were taken at 492 nm using 
 a microplate reader ( infinite m200 ) .
the minimum inhibitory concentrations 
 ( mic ) were defined as the lowest antifungal concentrations that caused 
 80% reduction in metabolic activity . for determining 
 the inhibition of biofilm formation , the medium was removed by aspiration 
 from cells that were actively growing in microtiter plates , and the 
 wells were washed twice with sterile pbs to remove nonadherent cells . 

fresh medium ( 100 l rpmi-1640 plus mops ) was then added back 
 to each well .
the 50% inhibitory concentration values ( ic50 ) for biofilm 
 formation inhibition were calculated using graphpad prism 5 .
the effects 
 of compounds on biofilm formation were confirmed by fluorescence microscopy 
 ( operetta , perkinelmer ) . after washing with pbs , yeasts were stained 
 with calcofluor white and held at 37 c for 10 min . to evaluate 
 the synergistic effects of compound 17 with amphotericin b ,
albicans cells were seeded in 96-well 
 plates and treated with different concentrations of test compounds 
 alone or in combination with amphotericin b in rpmi-1640 plus mops 
 medium at 37 c for 48 h. the viability of the yeast was measured 
 using the xtt assay , and the mic for growth was defined as the lowest 
 antifungal concentrations that caused 80% reduction in the 
 metabolic activity .
the interactions of test compounds with amphotericin 
 b were determined according to the resultant fici values .
fici values 
 were calculated as follows : fici = fica + ficb = ( mica  combination / mica  single ) 
 + ( micb  combination / micb  single ) . 

the interpretation of the fici data was determined as follows : 0.5 , 
 synergistic effect ; > 0.5 but < 4 , indifference ( no effect ) ; and 
 4 , antagonistic effect .
colorless , amorphous 
 solid ; uv ( meoh ) max ( log  ) 206 ( 4.31 ) nm ; 
 [ ]d25 + 3 ( c 0.24 , meoh ) ; h and c nmr ( see table 1 ) ; hresims m / z 625.3934 [ m + na ] ( calcd for c32h58o10na , 625.3928 ) .
colorless , amorphous 
 solid ; uv ( meoh ) max ( log  ) 206 ( 4.27 ) nm ; 
 [ ]d25 + 12 ( c 0.26 , meoh ) ; h and c nmr ( see table 1 ) ; hresims m / z 929.5474 [ m + na ] ( calcd for c46h82o17na , 929.5450 ) .
colorless , prism - shaped 
 crystals ; uv ( meoh ) max ( log  ) 206 ( 4.29 ) 
 nm ; [ ]d25 + 7 ( c 0.12 , meoh ) ; h and c nmr ( see table 1 ) ; hresims m / z 723.4667 [ m + na ] ( calcd for c38h68o11na , 723.4659 ) .
nmr spectra were obtained on a varian unity inova spectrometer ( 400 
 mhz for h and 100 mhz for c ) with a broad 
 band probe at 20  0.5 c using methanol - d4 ( cambridge isotope laboratories , inc . ) as the solvent . 

hresims spectra were collected on an agilent 6538 ultra high definition 
 accurate - mass q - tof system .
the lc - ms data were acquired on a shimadzu 
 uflc coupled to a quadrupole mass spectrometer using a phenomenex 
 kinetex c18 column ( 3.0 mm  75 mm , 2.6 m particle 
 size ) and mecn  h2o ( with 0.1% hcooh ) gradient ( 1:9 
 to 0:10 in 12 min ) followed by a 100% mecn wash .
column chromatography 
 was conducted using silica gel 60 ( 4063 m particle 
 size ) and sephadex lh-20 .
tlc silica gel 60 f254 plates 
 from emd chemicals inc . were used for tlc .
separations were also carried 
 out using a shimadzu hplc system equipped with lc-6ad solvent delivery 
 pumps coupled to an spd-10av uv  vis detector or a waters system 
 equipped with a 1525 binary hplc pump coupled to a 2998 pdad detector . 

both systems utilized phenomenex c18 columns ( 21.2  
 250 mm or 10  250 mm , 5 m particle size ) for preparative 
 runs .
x - ray diffraction data for compound 7 were collected 
 using a diffractometer with a bruker apex ccd area detector and graphite - monochromated 
 mo k radiation (  = 0.710  73  ) .
a 
 soil sample was collected from beneath a granite monument on the grounds 
 of one of the author s ( j.b.k . ) family property in buffalo 
 valley , oklahoma .
the sample was processed using the method described 
 by du et al . , and 51 fungal isolates were 
 purified .
the large - ribosomal - subunit internal transcribed spacer 1 ( its1 ) region 
 of the rdna gene was sequenced at the laboratory for genomics and 
 bioinformatics , ou health sciences center .
sequence data for the isolate 
 ( genbank accession km000126 ) was compared by blast analysis to sequences publicly 
 available through the ncbi database .
for the 1 l erlenmeyer 
 flask cultures , an 80 ml volume of cheerios breakfast cereal was added 
 to each flask , and the flasks were autoclaved using a solid - cycle 
 at 121 c for 30 min .
after the flasks were cooled to room temperature , 
 a 48 ml aliquot of autoclaved 0.3% sucrose with 0.005% chloramphenicol 
 was pipetted into the flasks to rehydrate the cereal .
one 5 
 mm  5 mm chunk of the fungal isolate grown on an agar plate 
 was added to each flask , and the cultures were incubated for 4 weeks .
when mycobags were used as the fermentation vessels , cheerios breakfast 
 cereal was preautoclaved using a solid - cycle at 121 c for 30 
 min . after cooling ,
a 500 ml volume of cheerios was added to each 
 bag , the openings of the bags were folded over once , the folds were 
 sealed with clamps , and bags were autoclaved using a liquid - cycle 
 at 121 c for 30 min .
eighteen 5 mm  5 mm chunks 
 of fungal culture grown on an agar plate were prepared and mixed with 
 the cereal .
the cereal was then rehydrated with 850 ml of an autoclaved 
 solution consisting of 0.3% sucrose with 0.005% chloramphenicol .
the 
 bags were placed flat to facilitate the formation of a monolayer of 
 cheerios across the bottom of the bag , and the cultures were incubated 
 for 4 weeks .
 one of the challenges presented by candida infections 
 is that many of the isolates encountered in the clinic produce biofilms , 
 which can decrease these pathogens susceptibilities to standard - of - care 
 antibiotic therapies . 
 inhibitors of fungal biofilm formation offer 
 a potential solution to counteracting some of the problems associated 
 with candida infections . a screening campaign utilizing 
 samples from our fungal extract library revealed that a bionectria 
 ochroleuca isolate cultured on cheerios breakfast cereal 
 produced metabolites that blocked the in vitro formation 
 of candida albicans biofilms . 
 a scale - up culture 
 of the fungus was undertaken using mycobags ( also known as mushroom 
 bags or spawn bags ) , which afforded four known [ tmc-151s c  
 f 
 ( 14 ) ] and three new [ bionectriols 
 b  d ( 57 ) ] polyketide glycosides . 
 
 all seven metabolites exhibited potent biofilm inhibition against c. albicans sc5314 , as well as exerted synergistic antifungal 
 activities in combination with amphotericin b. in this report , we 
 describe the structure determination of the new metabolites , as well 
 as compare the secondary metabolome profiles of fungi grown in flasks 
 and mycobags . 
 these studies demonstrate that mycobags offer a useful 
 alternative to flask - based cultures for the preparative production 
 of fungal secondary metabolites . 
Results and Discussion
Experimental Section
General Experimental
Procedures
Fungal
Isolate Procurement and Culture Conditions