Eriksen, Niels T.

Larsen, Poul

Nielsen, Per Halkjær

Nierychlo, Marta

English

Escherichia coli fermentations have been studied for decades, but most results are based on average measurements of the whole populations of cells, whilst averaged data can mask the distribution of activities at the sub-population or single-cell level. A population of genetically identical cells can exhibit different phenotypes under specific environmental conditions that show significant differences in physiological parameters from the population average. However, studies concerning segregation of populations into metabolically diversified subpopulations are scarce. Acetate is a product of Escherichia coli overflow metabolism when the bacteria are grown under aerobic conditions and glucose is present in excessive concentrations. Acetate accumulation is of the utmost importance in batch fermentation processes as it is an undesirable byproduct that negatively affects growth, physiology, and performance of Escherichia coli. An insight into glucose and acetate fate on the level of individual cell can provide the type of information which are valuable for the understanding of bacterial metabolism in fermentation process and can shed more light on the differentiation of isogenic fermenting populations into subpopulations expressing metabolically different profiles. The goal of this study was to observe and quantify in situ metabolic response of Escherichia coli at single-cell level and determine activity distribution of glucose and acetate uptake during batch fermentation processes. Pure culture of Escherichia coli MG1655 was used to investigate glucose and acetate metabolism at single-cell level during different stages of glucose batch fermentation process. Uptake of the substrates was observed and measured in situ by quantitative microautoradiography. Sub-populations of Escherichia coli cells expressing different activity levels for the uptake of glucose or acetate were observed. The distribution of these uptake activities changed along the batch fermentation process. The results based on the observation of single cells indicate that heterogeneity exists within bacterial populations and is a result of metabolic diversification of individual cells.</p

    Aalborg UniversitetSingle-cell level based approach to investigate bacterial metabolism during batchindustrial fermentationNierychlo, Marta; Larsen, Poul; Eriksen, Niels T.; Nielsen, Per HalkjærPublication date:2012Document VersionEarly version, also known as pre-printLink to publication from Aalborg UniversityCitation 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.General rightsCopyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright ownersand it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights.            ? Users may download and print one copy of any publication from the public portal for the purpose of private study or research.            ? You may not further distribute the material or use it for any profit-making activity or commercial gain            ? You may freely distribute the URL identifying the publication in the public portal ?Take down policyIf you believe that this document breaches copyright please contact us at vbn@aub.aau.dk providing details, and we will remove access tothe work immediately and investigate your claim.Downloaded from vbn.aau.dk on: December 26, 2020Single-­‐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 	  	  	  	  	  	  starvation0	  10	  20	  30	  40	  50	  60	  0	   1	   2	   3	   4	   5	   6	   7	   8	   9	   10	  %	  of	  total	  popula4on	  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	  number	  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	  

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

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

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