Aalborg Universitet

Single-cell level based approach to investigate bacterial metabolism during batchindustrial fermentation

Nierychlo, Marta; Larsen, Poul; Eriksen, Niels T.; Nielsen, Per Halkjær

Publication date:2012

Document VersionEarly version, also known as pre-print

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Citation for published version (APA):Nierychlo, M., Larsen, P., Eriksen, N. T., & Nielsen, P. H. (2012). Single-cell level based approach to investigatebacterial metabolism during batch industrial fermentation. Poster presented at 14th International Symposium onMicrobial Ecology, Copenhagen, Denmark.

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Single-­‐cell  level  based  approach  to  inves4gate  bacterial  metabolism  during  batch  industrial  fermenta4on    

                                                       

                                                       

Most   of   the   data   from   Escherichia   coli   fermenta.ons   are   based   on   the  average   measurement   of   the   whole   popula.on,   which   can   mask   the  distribu.on   of   the   ac.vity   on   the   sub-­‐popula.on   level.   It   is   known   that   a  popula.on   of   gene.cally   iden.cal   cells   can   exhibit   different   phenotypes  under   specific   environmental   condi.ons,   however,   studies   concerning   the  segrega.on   of   star.ng   popula.ons   into   metabolically   diversified   sub-­‐popula.ons  are  scarce.    Acetate   is   a   product   of   E.coli   overflow  metabolism  when   cells   are   grown  under  aerobic  condi.ons  in  the  presence  of  excess  glucose.  Minimizing  the  accumula.on  of   acetate   is   cri.cal   in   batch   fermenta.on  processes   as   this  undesirable  by-­‐product  has  a  nega.ve  affect  on  the  growth,  physiology,  and  performance  of  E.coli.    Monitoring   the   fate   of   glucose   and   acetate   on   the   single-­‐cell   level   will  provide   valuable   insight   into   bacterial   metabolism   in   the   fermenta.on  process;  shedding  more   light  on  the  differen.a.on  of   isogenic  popula.ons  into  sub-­‐popula.ons  that  exhibit  different  metabolic  profiles.    

I  n  t  r  o  d  u  c  t  i  o  n  

                                                       

                                                       

                                                       

                                                       

R  e  s  u  l  t  s  

                                                       

                                                       

M  e  t  h  o  d  s  

mni@bio.aau.dk  

Marta  Nierychlo,  Poul  Larsen,  Niels  T.  Eriksen,  Per  H.  Nielsen  Department  of  Biotechnology,  Chemistry  and  Environmental  Engineering,  Aalborg  University,  Aalborg,  Denmark  

                                                       

O  b  j  e  c  t  i  v  e  s  

                                                       

C  o  n  c  l  u  s  i  o  n  s  

v Heterogeneity  in  the  uptake  of  both  glucose  and  acetate  exists  in  batch  industrial  fermenta4ons  and  is  present  at  each  stage  of  the  process  

v Fermen4ng  E.coli  popula4ons  differen4ate  into  sub-­‐popula4ons  of  cells  exhibi4ng   different   metabolic   strategies   and   different   levels   of  metabolic  ac4vity  

v The   consump4on   of   acetate   during   batch   fermenta4on  was   shown   to  start  surprisingly  early  in  the  exponen4al  phase.  This  phenomenon  has  not  been  demonstrated  before  as  previous  studies,  based  on  popula4on  average  measurements,   indicated   that   acetate   uptake   starts   closer   to  glucose  deple4on.  

Batch   fermenta4ons   (MOPS   defined   mineral   media     +   2.5   g/L  glucose)   were   performed   in   order   to   examine   bacterial  metabolism   on   the   consecu.ve   stages   of   the   fermenta.on  process.  

Samples  for  metabolic  ac.vity  determina.on  were  taken  at  different  .me  points  during  the  fermenta.on  process.  

Fermenta.on   1  

Cells  were  stained  with  fluorescent  dye  (DAPI  or  PI)  and  observed  under  the  microscope.    MAR  signal  was  quan.fied  with  the  help  of  freeware  ImageJ.  Substrate   uptake  was   evaluated   by   enumera.ng   the   number   of  silver  grains  within  the  defined  boundary  around  individual  E.coli  cells.      

Microscopy  &  Image  Analysis   3  

Incubation with 3H-acetate / 3H-glucose

The  uptake  of  acetate/glucose  at  the  single  cell  level   was   inves.gated   in   situ   by   means   of    Microautoradiography  (MAR).        

Substrate  uptake  determina.on   2  

A  pure  culture  of  E.coli  MG1655  was  used  to  inves.gate  in  situ  glucose  and  acetate  metabolism  at  the  single-­‐cell  level.  

Sample  no. Sample  description1            exponential  glucose  uptake2            cease  of  exponential  growth3            glucose  exhaustion4            beginning  of  acetate  uptake5            advanced  acetate  consumption6            starvation

0  

10  

20  

30  

40  

50  

60  

0   1   2   3   4   5   6   7   8   9   10  

%  of  total  pop

ula4

on  

No.  of  grains  per  cell  (uptake  ac4vity)  

Distribu4on  of    glucose  uptake  ac4vity    

in  E.coli  batch  fermenta4on  

exponen.al   sta.onary  

•  As   expected,   the   cells   in  exponen.al   phase   exhibit  much   higher   metabolic  ac.vity   than   the   cells   in  sta.onary  phase    

•  A   significant   number   of  E.coli   cells   retain   metabolic  ac.vity  during  the  sta.onary  phase  

•  There   are   a   number  of   cells  in   exponen.al   phase,  which  are   thought   to   be   ac.ve,  that   do   not   show   substrate  uptake  

•  Acetate   consump.on   starts  early   in   the   course   of   batch  fermenta.on  

•  Only   a   sub-­‐popula.on   of  E.coli   cells   are   able   to   take  up  acetate  

•  The   number   of   cells   taking  u p   a c e t a t e   d u r i n g  f e rmen ta.on   change s  depending   on   the   stage   of  the  process  

0  

10  

20  

30  

40  

50  

60  

70  

80  

90  

100  

0   1   2   3   4  

%  of  total  cell  num

ber  

No.  of  silver  grains  per  cell  (uptake  ac4vity)  

Distribu4on  of    acetate  uptake  ac4vity    

in  E.coli  batch  fermenta4on  

3.5  h   5.5  h   6.25  h   8.25  h   10.0  h   26.0  h  

1   2   3   4   5   6  

v To  observe  and  quan.fy  the  in  situ  metabolism  of  glucose  and  acetate  by  E.coli  during  batch  fermenta.on  at  the  single  cell  level  

v To   check   if   bacterial   sub-­‐popula.ons   exist,   that   exhibit   different  metabolic  strategies    towards  the  inves.gated  substrates  

v To   find   out   when   the   acetate   uptake   starts   in   the   batch   fermenta.on  process  and  if  all  cells  equally  contribute  to  its  consump.on