Production of Single Cell Protein from Residual Streams from 2 nd Generation Bioethanol Production

      MASTER  OF  SCIENCE  THESIS  WITHIN  BIOTECHNOLOGY     Production  of  Single  Cell  Protein  from  Residual   Streams  from  2nd  Generati...
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MASTER  OF  SCIENCE  THESIS  WITHIN  BIOTECHNOLOGY    

Production  of  Single  Cell  Protein  from  Residual   Streams  from  2nd  Generation  Bioethanol  Production     Amanda  Steen  

  Performed  at  SP  Processum  AB   Örnsköldsvik,  Sweden   Spring  2014     Supervisor:  Björn  Alriksson,  SP  Processum  AB   Examiner:  Gen  Larsson,  KTH           School  of  Biotechnology   Division  of  Bioprocess  Technology   The  Royal  Institute  of  Technology,  KTH   Stockholm,  Sweden,  2014  

Production  of  Single  Cell  Protein  from  Residual  Streams     from  2nd  Generation  Bioethanol  Production    

Abstract   The   demand   of   food,   and   especially   protein,   is   increasing   along   with   the   increase   of   the   global   human  population.  Fish  is  an  important  source  of  protein  for  the  global  population.  The  demand   for  aquatic  food  is  projected  to  increase  with  about  20%  between  the  year  2010  and  2020.  This   increase   has   to   be   met   by   increased   aquaculture   production.   Consequently,   there   is   an   increasing   demand   for   fish   feed.   The   favoured   protein   source   in   fish   feed   is   fishmeal.   The   availability   of   fishmeal   is   however   predicted   to   decrease.   Therefore,   there   is   an   increasing   demand   for   alternative   high-­‐quality   protein   sources   for   fish   feed.   Soybean   meal   is   the   most   frequently   used   alternative   source   for   fish   feed   today.   However,   vegetable   protein   sources   are   associated   with   challenges,   such   as   antinutritional   substances   and   unfavourable   amino   acid   composition.   Single   cell   protein   (SCP)   is   another   alternative   protein   source   that   has   many   benefits,  such  as  a  fast  production  and  a  favourable  amino  acid  profile.  In  addition,  SCP  can  be   produced  from  different  residual  streams  derived  from  industry.  This   provides  the  possibility  to   have  a  cheap  production  from  renewable  and  sustainable  feedstocks.     In   this   master   thesis   project   the   potential   to   produce   SCP   from   residual   streams   from   the   2nd   generation   bioethanol   production   has   been   investigated.   Three   different   residual   streams   based   on   lignocellulosic   material   (prehydrolysate   and   stillage   of   wheat   straw,   and   prehydrolysate   of   spruce)  were  utilised  and  four  different  microorganisms  were  evaluated  (Paecilomyces  variotii,   Cunninghamella  echinulata,  Mortierella  isabellina,  and  Yarrowia  lipolytica).  Pilot-­‐scale  cultivation   of   P.   variotii   on   prehydrolysate   of   wheat   straw   and   on   detoxified   prehydrolysate   of   spruce   showed  promising  results  with  biomass  concentrations  of  8-­‐10  g/L  and  with  a  protein  content  of   around  50%.  In  addition,  the  biomass  consisted  of  high  levels  of  β-­‐glucans,  about  20%.  β-­‐glucans   are   an   interesting   molecule   that   increasingly   is   being   supplied   to   fish   feed   due   to   their   immunostimulatory  effect.  The  high  β-­‐glucan  content  could  potentially  increase  the  value  of  the   SCP   as   an   ingredient   in   fish   feed.   Y.  lipolytica  grew   well   on   stillage   of   wheat   straw   and   reached   a   biomass   concentration   of   15   g/L   with   a   protein   content   of   over   50%   in   a   pilot-­‐scale   experiment.   An   interesting   finding   was   the   utilisation   of   uncharacterised   carbon   sources   within   the   prehydrolysate   and   stillage   of   wheat   straw.   This   indicates   that   the   microorganisms,   and   especially  Y.  lipolytica,  were  able  to  utilise  a  broad  range  of  the  carbon  sources  available  within   the   residual   streams.   This   study   shows   that   the   utilisation   of   residual   streams   from   the   2nd   generation   bioethanol   production   is   an   interesting   and   potential   substrate   for   large-­‐scale   production  of  SCP,  which  warrants  further  studies.            

 

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Production  of  Single  Cell  Protein  from  Residual  Streams     from  2nd  Generation  Bioethanol  Production    

Sammanfattning   Världens   befolkning   ökar   och   med   den   ökar   också   behovet   av   mat.   Fisk   är   en   viktig   proteinkälla   för  en  stor  del  av  jordens  befolkning.  Efterfrågan  på  fisk  förväntas  öka  med  nästan  20%  mellan   år   2010   och   2020.   Denna   ökning   måste   komma   från   produktion   i   fiskodlingar,   vilket   i   sin   tur   innebär  en  ökad  efterfrågan  på  fiskfoder.  Fiskmjöl  är  generellt  den  mest  använda  proteinkällan   för  fiskfoder,  men  tillgången  av  fiskmjöl  förväntas  minska  kommande  år.  Följaktligen  finns  det   ett   ökande   behov   av   alternativa   högkvalitativa   proteinkällor   som   ersättningsprodukt   för   fiskmjöl.   Idag   är   sojamjöl   den   vanligaste   alternativa   proteinkällan   i   fiskfoder.   Proteinkällor   i   form  av  grödor  och  andra  växter  är  dock  förknippat  med  vissa  nackdelar,  såsom  antinutrionella   ämnen   och   en   ofördelaktig   aminosyraprofil.   Single   cell   protein   (SCP)   är   en   annan   intressant   alternativ   proteinkälla.   SCP   har   många   fördelar   såsom   en   snabb   proteinproduktion   och   en   fördelaktig   aminosyraprofil.   SCP   kan   dessutom   produceras   från   restströmmar   från   olika   industrier,   vilket   ger   möjligheten   till   en   billig   produktion   från   förnyelsebara   och   hållbara   råmaterial.   I   detta   examensarbete   har   potentialen   att   producera   SCP   från   restströmmar   från   2:a   generationens   bioetanolproduktion   undersökts.   Tre   olika   restströmmar   från   bioetanolproduktion   från   lignocellulosa   (förhydrolysat   och   drank   från   vetehalm,   samt   förhydrolysat  från  gran)  och  fyra  olika  mikroorganismer  (Paecilomyces  variotii,  Cunninghamella   echinulata,   Mortierella   isabellina   och   Yarrowia   lipolytica)   utvärderades.   Odling   av   P.   variotii   i   pilotskala  på   förhydrolysat   från   vetehalm   och   detoxifierat   förhydrolysat   från   gran   gav   lovande   resultat  och  resulterade  i  biomassakoncentrationer  på  8-­‐10  g/L  med  ett  proteininnehåll  på  cirka   50%.   Dessutom   bestod   biomassan   av   cirka   20%   β-­‐glukan.   β-­‐glukan   är   en   grupp   intressanta   molekyler   med   immunostimulerande   egenskaper   vilka   används   i   allt   större   utsträckning   inom   fiskfoderindustrin.   Ett   högt   innehåll   av   β-­‐glukan   kan   potentiellt   öka   värdet   på   SCP   som   en   ingrediens   i   fiskfoder.   Även   Y.   lipolytica   växte   bra   på   drank   från   vetehalm   och   gav   biomassakoncentrationer   på   15   g/L   med   ett   proteininnehåll   på   över   50%   i   odling   i   pilotskala.   Ett   intressant   resultat   från   studien   är   att   mikroorganismerna   använde   andra   kolkällor,   än   de   som   analyserades   och   identifierades   i   restströmmarna   från   vetehalm.   Detta   visar   att   mikroorganismerna,  och  särskilt  Y.  lipolytica,  har  möjligheten  att  i  stor  utsträckning  använda  de   källor   av   kol   som   finns   närvarande   i   de   undersökta   restströmmarna.   Detta   arbete   visar   att   restströmmar   från   produktion   av   2:a   generationens   bioetanol   är   intressanta   och   lovande   som   substrat   för   storskalig   produktion   av   SCP,   och   att   vidare   studier   inom   detta   område   bör   genomföras.    

 

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Production  of  Single  Cell  Protein  from  Residual  Streams     from  2nd  Generation  Bioethanol  Production    

Table  of  contents   Abstract  ...............................................................................................................................................  ii   Sammanfattning  .............................................................................................................................  iii   1.  Introduction  ..................................................................................................................................  2   2.  Background  ...................................................................................................................................  5   2.1  Single  cell  protein  and  single  cell  oil  ............................................................................................  5   2.2  Microorganisms  for  single  cell  protein  and  single  cell  oil  production  .............................  9   2.3  Bioethanol  production  from  lignocellulose  ............................................................................  11   3.  Materials  and  methods  ..........................................................................................................  15   3.1  Microorganisms  and  residual  streams  .....................................................................................  15   3.2  Preparation  of  residual  streams  .................................................................................................  16   3.3  Initial  screening  experiments  with  prehydrolysate  and  stillage  of  wheat  straw  ......  16   3.4  Detoxification  experiments  with  prehydrolysate  and  stillage  of  wheat  straw  ...........  17   3.5  Initial  screening  and  detoxification  experiment  with  prehydrolysate  of  spruce  ......  18   3.6  Multifermenter  experiment  with  P.  variotii  on  prehydrolysate  and  stillage  of  wheat   straw  ............................................................................................................................................................  19   3.7  Pilot  scale  experiments  ..................................................................................................................  22   3.8  Chemical  analyses  ............................................................................................................................  30  

4.  Results  .........................................................................................................................................  32   4.1  Concentrations  of  monosaccharides,  aliphatic  acids,  and  ethanol  in  the  residual   streams  .......................................................................................................................................................  32   4.2  Initial  screening  experiments  with  prehydrolysate  and  stillage  of  wheat  straw  ......  33   4.3  Detoxification  experiments  with  prehydrolysate  and  stillage  of  wheat  straw  ...........  37   4.4  Initial  screening  and  detoxification  experiments  with  prehydrolysate  of  spruce  ....  40   4.5  Multifermenter  experiment  with  prehydrolysate  and  stillage  of  wheat  straw  ..........  45   4.6  Pilot  scale  experiments  ..................................................................................................................  46  

5.  Discussion  ..................................................................................................................................  57   6.  Future  work  ...............................................................................................................................  66   Acknowledgement  .......................................................................................................................  67   References  ......................................................................................................................................  68   Appendix  I  .......................................................................................................................................  73   Appendix  II  .....................................................................................................................................  75   Appendix  III  ....................................................................................................................................  76   Appendix  IV  ....................................................................................................................................  77   Appendix  V  ......................................................................................................................................  79   Appendix  VI  ....................................................................................................................................  81    

 

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Production  of  Single  Cell  Protein  from  Residual  Streams     from  2nd  Generation  Bioethanol  Production    

1.  Introduction   In  a  time  when  the  global  human  population  is  increasing,  the  demand  on  food,  and  especially   protein  is  a  hot  topic  given  high  focus.  In  the  year  2050  the  global  human  population  is  expected   to   have   reached   9   billion   (The   World   Bank,   2013).   Fish   is   an   important   source   of   nutritious   food   and   protein   for   many   of   the   world’s   humans.   In   the   year   of   2010   the   level   of   aquatic   food   consumption   was   128   million   tonnes.   An   increase   of   23   million   tonnes   is   estimated   to   be   required   until   2020,   if   the   current   level   of   per-­‐capita   consumption   of   aquatic   foods   is   to   be   maintained.   Today,   the   majority   of   the   marine   fish   stocks   are   exploited,   overexploited,   or   depleted   and   there   is   no   room   for   further   expansion.   The   decreasing   fish   stocks   of   the   oceans   imply   that   the   predicted   increased   demand   of   aquatic   food   will   have   to   be   met   by   production   through  aquacultures  and  not  from  captured  fish  (FAO  Fisheries  and  Aquaculture  Department,   2012).     Today,   almost   50%   of   the   global   food   fish   supply   comes   from   aquaculture   production   (FAO   Fisheries   and   Aquaculture   Department,   2012).   During   the   last   three   decades   the   aquaculture   production  has  increased  from  5  million  to  63  million  tonnes.  It  is  expected  that  the  aquaculture   production   further   will   expand   substantially,   to   almost   94   million   tonnes  by   the   year   2030   (The   World   Bank,   2013).   Currently   the   aquaculture   sector   grows   with   an   average   rate   of   8   to   10   percent   per   year.   Almost   50%   of   the   global   aquaculture   productions   are   reliant   of   addition   of   fish   feed.   In   order   to   keep   up   with   the   increasing   aquaculture   production,   the   supply   of   feed   sources  (e.g.  protein)  will  have  to  grow  with  a  similar  rate  (Tacon  et  al.,  2011).  Fishmeal  is  the   favoured  protein  source  for  many  aquaculture  species.  However,  it  is  predicted  that  the  use  of   fishmeal  in  fish  feed  will  decrease  with  about  7%  until  2015  (compared  to  2012),  partly  due  to   reduction   in   the   supplies   of   caught   fish   due   to   tighter   quotas,   stricter   control   of   unregulated   fishing,   and   enlarged   utilisation   of   more   low-­‐cost   dietary   fishmeal   substitutes   (FAO   Fisheries   and   Aquaculture   Department,   2012).   As   a   consequence,   there   is   an   increasing   demand   for   alternative  high-­‐quality  protein  sources  for  fish  feed.     Vegetable  protein  sources  are  today  commonly  used  in  fish  feed.  The  most  frequent  alternative   protein   source   used   in   aquaculture   feed   is   soybean   meal   (FAO   Fisheries   and   Aquaculture   Department,   2012).   Feed   of   plant   origin   can   however   only   be   used   to   a   limited   extent   due   to   their  amino  acid  composition  being  different  compared  to  that  of  fishmeal  protein.  In  addition,   plant-­‐derived   materials   can   contain   various   antinutritional   substances.   These   substances   can   have  a  negative  effect  on  the  health  and  productivity  of  animals  (Francis  et  al.,  2001).       Another   possible   alternative   protein   source   is   single   cell   protein   (SCP).   SCP   consists   of   microorganisms  such  as  filamentous  fungi,  yeast,  algae,  and  bacteria  that  are  rich  in  protein.  SCP   has   many   benefits.   It   is   a   very   fast   way   of   producing   protein   compared   to   the   production   of   protein   through   cultivation   of   agricultural   crops   or   animal   farming.   The   amino   acid   profile   of   many   SCP   is   favourable   and   very   similar   to   that   of   fishmeal   (Nitayavardhana   et   al.,   2013)(Alriksson   et   al.,   2014)(UniBio   A/S,   2014).   SCP   can   be   produced   from   residual   streams   from   different   industries   giving   the   possibility   of   a   cheap   production   (Almeida   e   Silva   et   al.,   1995)(Alriksson  et  al.,  2014).  In  addition,  SCP  production  can  be  performed  in  bioreactors  and   does   not   hold   up   agricultural   land.   Production   of   SCP   may   very   well   fit   into   the   request   of   a   sustainable   high-­‐quality   alternative   to   fishmeal   since   the   production   can   be   performed   using    

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Production  of  Single  Cell  Protein  from  Residual  Streams     from  2nd  Generation  Bioethanol  Production     renewable  and  sustainable  feedstocks  such  as  residual  streams  from  2nd  generation  bioethanol   production.     The   2nd   generation   bioethanol   production   (i.e.   production   from   lignocellulosic   materials)   is   predicted   to   increase   in   the   future,   resulting   in   large   volumes   of   residual   and   waste   streams   (Limayem   and   Ricke,   2012)(Nitayavardhana   and   Khanal,   2012).   These   residual   streams   are   commonly   considered   to   be   used   as   substrates   for   biogas   production   (Ekman   et   al.,   2013)   (SEKAB,  2014).  SCP  production  is  an  interesting  alternative  to  biogas  production,  possibly  with  a   higher   economic   value.   However,   the   usage   of   residual   streams   from   the   2nd   generation   bioethanol   is   associated   with   several   challenges.   The   complexity   of   the   residual   streams   is   usually   quite   high   with   different   types   of   sugars   and   degradation   products   of   lignocellulose.   Some   of   the   degradation   products   can   inhibit   the   growth   of   microorganism   used   for   SCP   production.  It  is  essential  to  find  microorganisms  suitable  for  the  specific  residual  stream  to  be   used,  microorganisms   that   are   able   to   utilise  as  much   as   possible   of  the  different  carbon  sources   present.   In   addition,   counteractions   regarding   the   inhibitors   present   in   the   residual   streams   have  to  be  considered.   Except  protein,  some  microorganisms  can  also  produce  microbial  oils  and  lipids,  referred  to  as   single   cell   oil   (SCO).   As   well   as   there   is   a   need   of   alternative   protein   sources   in   fish   feed,   is   there   also   a   need   for   alternative   lipid   sources   that   can   substitute   fish   oils   (FAO   Fisheries   and   Aquaculture   Department,   2012).   Analogously   to   SCP   and   fishmeal,   SCO   production   offers   an   interesting  alternative  to  fish  oils.     The   aim   with   this   master   thesis   project   has   been   to   investigate   the   potential   of   producing   SCP   and  SCO  from  different  residual  streams,  coming  from  the  lignocellulosic  ethanol  industry.  Three   different  residual  streams  from  bioethanol  production  from  agriculture-­‐  and  forestry  materials   have  been  investigated  as  carbon  source  for  production  of  SCP  or  SCO:  i)  prehydrolysate  from  a   bioethanol  process  based  on  wheat  straw,  ii)  stillage  from  a  bioethanol  process  based  on  wheat   straw,  iii)  prehydrolysate  from  a  bioethanol  process  based  on  spruce  wood  chips.  The  different   microorganisms   investigated   were   three   filamentous   fungi,   Paecilomyces   variotii,   Cunninghamella   echinulata,   Mortierella   isabellina,   and   the   yeast   Yarrowia   lipolytica.   Figure   1   displays   a   typical   process   scheme   for   the   production   of   ethanol   from   lignocellulosic   materials.   The  residual  streams  used  as  substrates  in  this  study  are  shown  as  the  red  arrows  in  the  figure.  

 

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Production  of  Single  Cell  Protein  from  Residual  Streams     from  2nd  Generation  Bioethanol  Production      

Lignocellulose' Wheat'straw' or'spruce'

Pre8 treatment'

Separa"on' solid/liquid'

Enzyma"c' hydrolysis'

Fermenta"on' SFF'

Dis"lla"on'

S"llage'

Prehydrolysate' liquid'frac"on'

Separa"on'' solid/liquid' SCP' produc"on'

Combined'heat8' and'power'plant'

S"llage'' liquid'frac"on'

SCP' produc"on'

Dissolved' material'

Solid'fuel'

Energy'

Ethanol'

Biogas'

Anaerobic' treatment'

Biofuel'

  Figure  1  –  Outline  for  a  typical  process  scheme  for  the  production  of  ethanol  from  lignocellulosic   materials.   The   red   arrows   indicate   the   residual   streams   used   as   substrates   in   this   study   (modified   from  Galbe  and  Zacchi,  2012).  

   

 

 

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Production  of  Single  Cell  Protein  from  Residual  Streams     from  2nd  Generation  Bioethanol  Production    

2.  Background   The  following  sections  will  give  a  background  regarding  the  concept  of  SCP,  and  also  SCO,  along   with  a  short  description  about  the  history  of  SCP  production,  and  microorganisms  used  for  SCP   and   SCO   production.   Information   about   the   usage   of   fish   feed   is   also   provided,   together   with   knowledge  regarding  possible  toxins  and  immunostimulants  in  microorganisms  for  SCP  and  SCO   production.  Furthermore,  a  general  description  of  the  process  of  bioethanol  production,  yielding   the   residual   streams   like   the   ones   used   in   this   study,   is   given,   including   information   regarding   the   biomass   used,   inhibitors   that   can   be   produced   during   the   process,   and   possible   counteractions  that  can  be  taken  to  overcome  the  problem  with  inhibitors.  

2.1  Single  cell  protein  and  single  cell  oil   SCP  is  the  definition  for  dried  cells  originated  from  single-­‐celled  organisms  intended  to  be  used   as  a  protein  source  in  human  foods  or  animal  feeds.  The  type  of  microorganisms  used  includes   bacteria,  algae,  yeasts,  molds,  and  other  fungi  (Litchfield,  1983)(Nasseri  et  al.,  2011).  Despite  the   name,  SCP,  the  microbial  cells  do  not  exclusively  consist  of  protein.  Microbial  cells  also  consist  of   lipids,  carbohydrates,  vitamins,  minerals,  and  nucleic  acids  (Litchfield,  1983).     The  interest  in  SCP  started  already  some  time  before  World  War  I  (Ugalde  and  Castrillo,  2002).   During   the   World   War   I   Germany   tried   to   supplement   their   protein   supply   in   animal   feed   by   using  Baker’s  yeast.  They  managed  to  replace  as  much  as  half  of  all  the  protein  sources  imported   at   that   time   with   yeast   (Ugalde   and   Castrillo,   2002).   The   yeast   was   cultivated   on   molasses   as   carbon   source   and   ammonium   salts   were   used   as   nitrogen   source   (Litchfield,   1983).   After   the   end  of  the  World  War  I  the  interest  in  yeast  as  fodder  declined  but  arose  again  when  World  War   II  started.  At  this  point  yeast  had  been  included  into  the  army  diets,  and  after  some  time  also  into   the   diets   of   civilians.   However,   the   high   ambition   to   produce   more   than   100   000   tonnes   of   yeast   per   year   was   by   far   never   reached   (Ugalde   and   Castrillo,   2002).   The   yeast   of   interest   was   Candida  utilis  (Torula   yeast)   and   it   was   cultivated   on   sulphite   waste   liquor   from   the   pulp   and   paper   industries   and   on   wood   sugar   derived   from   acid   hydrolysis   of   wood   (Litchfield,   1983).   The  production  of  Torula  yeast  continued  after  the  World  War  II  in  the  United  States  as  part  of  a   larger  program  for  utilisation  of  natural  sources  for  fodder  (Ugalde  and  Castrillo,  2002).  In  the   early  60’s  various  companies  started  to  investigate  the  possibility  to  produce  SCP  that  could  be   used   as   protein   source,   as   a   response   to   the   concept   of   the   protein   gap,   which   had   been   brought   forward   by   The   Food   and   Agriculture   Organisation   of   the   United   Nations   (FAO).   Between   the   mid  60’s  and  the  80’s  the  SCP  industry  looked  very  promising  but  due  to  technical  and  political   developments  in  the  80’s  the  expansion  levelled  off.  Instead  of  an  increased  SCP  production  the   agricultural   production   increased   as   a   result   of   improved   production   and   distribution   knowledge.  Many  of  the  processes  for  SCP  were  ceased  as  a  direct  result  of  being  outcompeted   by  the  cheap  agricultural  crops.  Although,  a  very  successful  example  of  a  SCP  process,  that  has   taken  the  step  into  being  a  commercial  product,  is  the  production  of  Fusarium  venenatum,  which   is   sold   under   the   trademark   QuornTM.   QuornTM   constitute   a   fungal-­‐based   protein   source   produced  for  human  consumption  (Ugalde  and  Castrillo,  2002).   SCO   refers   to   microbial   oils   or   lipids   synthesised   by   microorganisms   (Ratledge,   2004)(Zeng   et   al.,   2013).   Some   microorganisms,   oleaginous   microorganisms,   are   able   to   accumulate   lipids   up    

5  

Production  of  Single  Cell  Protein  from  Residual  Streams     from  2nd  Generation  Bioethanol  Production     to  levels  of  20-­‐80%  of  their  cell  dry  weight  (Zeng,  et  al.,  2013).  All  living  organisms  synthesise   lipids  for  membranes  and  other  structures  but  these  lipids  corresponds  to  a  relatively  low  share   of   the   cell   weight.   Some   microorganisms   can   however   also   produce   and   accumulate   lipids   as   reserve  storage  and  these  microorganisms  are  typically  useful  for  production  of  SCO.  This  type   of  accumulation  is  found  in  some  yeasts  and  fungi,  along  with  a  small  number  of  algae  (Ratledge,   2004).   A  requirement  for  SCO  accumulation  is  a  cultivation  medium  with  an  excess  carbon  source  and   limiting   nitrogen   source.   The   accumulation   of   SCO   starts   first   when   the   nitrogen   is   depleted   (Ratledge,   2004).   The   excess   carbon   source   can   after   depletion   of   nitrogen   continue   to   be   assimilated  and  is  then  directed  into  lipid  synthesis,  thereby  building  up  triacylglycerols  in  the   form   of   small   oil   droplets.   The   capability   to   accumulate   oil   cannot   only   be   explained   by   the   fatty   acid   biosynthesis.   This   conclusion   is   based   on   that   non-­‐oleaginous   species   do   not   accumulate   any   oil   if   placed   in   a   nitrogen-­‐limited   medium.   Oleaginous   microorganisms   have   two   features   making  the  lipid  accumulation  possible.  First,  they  are  able  to  continuously  produce  acetyl-­‐CoA   directly   in   the   cytosol   (acetyl-­‐CoA   being   an   important   precursor   for   fatty   acid   synthase).   Secondly,  they  are  able  to  produce  a  sufficient  amount  of  NADPH,  which  is  an  essential  reductant   used  in  fatty  acid  biosynthesis  (Ratledge  2004).  The  acetyl-­‐CoA  is  formed  from  citrate  and  CoA   using  ATP  by  the  enzyme  ATP:citrate  lyase  (Reaction  [R1]),  which  does  not  seem  to  be  present   in  non-­‐oleaginous  microorganisms  (Ratledge,  2004).     𝐶𝑖𝑡𝑟𝑎𝑡𝑒 + 𝐶𝑜𝐴 + 𝐴𝑇𝑃

!"#:!"#$%!"  !"#

%$𝑎𝑐𝑒𝑡𝑦𝑙 − 𝐶𝑜𝐴 + 𝑜𝑥𝑎𝑙𝑜𝑎𝑐𝑒𝑡𝑎𝑡𝑒 + 𝐴𝐷𝑃 + 𝑃!    

[R1]  

2.1.1  Fish  feed  from  single  cell  protein   SCP   is   a   potential   protein   source   for   use   in   fish   feed,   as   well   as   SCO   is   a   potential   lipid   source.   Today,   the   search   for   alternative   protein   sources   for   fish   feed   is   a   hot   topic   since   the   use   of   fishmeal   is   predicted   to   decline   while   the   aquaculture   production   at   the   same   time   increases   (FAO  Fisheries  and  Aquaculture  Department,  2012).   Feed   used   for   aquaculture   production   is   categorised   into   three   main   groups:   animal   nutrient   sources,   plant   nutrient   sources,   and   microbial   nutrient   sources.   Animal   nutrient   sources   include   both   aquatic   and   terrestrial   animals.   Among   the   microbial   nutrient   sources   are   algae,   yeasts,   fungi,   bacteria   and/or   microbial   SCP   sources.   Today,   the   only   microbial   derived   feed   source   available  in  commercial  quantity  is  yeast-­‐derived  products,  such  as  brewer’s  yeast  and  extracted   fermented  yeast  products  (FAO  Fisheries  and  Aquaculture  Department,  2012).   The   most   common   alternative   protein   source   used   today   is   soybean   meal,   23%   (by   weight)   of   the   total   compounds   in   fish   feeds   were   made   out   of   soybean   meal   in   2008   (FAO   Fisheries   and   Aquaculture   Department,   2012).   Feed   with   plant   origin   can   however   only   be   used   to   a   limited   extent   due   to   their   amino   acid   composition   being   different   compared   to   fishmeal   protein.   In   addition,   plant-­‐derived   materials   contain   various   antinutritional   substances,   substances   which   interfere   with   the   utilization   of   feed   and   that   affect   the   health   and   production   of   animals   (Francis  et  al.,  2001).      

 

6  

Production  of  Single  Cell  Protein  from  Residual  Streams     from  2nd  Generation  Bioethanol  Production     As  described  in  the  introduction  (Section  1)  the  benefits  of  SCP  as  an  alternative  protein  source   in  fish  feed  are  many.  Microorganisms  have  a  short  generation  time  and  grow  much  faster  than   many   of   the   agricultural   or   animal   protein   sources.   Another   benefit   with   using   SCP   is   the   possibility   to   have   the   production   based   on   industrial   residual   and   waste   streams,   an   aspect   which  is  attractive  from  both  an  economical  and  environmental  point  of  view.     Today   there   is   a   great   need   for   a   sustainable   fishery   industry   (FAO   Fisheries   and   Aquaculture   Department,   2012).   The   choice   of   ingredients   for   aquaculture   feed   should   not   only   be   based   upon   nutrient   level,   digestibility,   and   cost,   but   also   upon   the   sustainability   and   environmental   impact  of  the  production.  Such  criteria  come  somewhat  hand  in  hand  since  using  a  high-­‐quality   feed   source   will   contribute   to   less   nutrient   loss   and   feed   wastage,   which   will   minimize   the   negative  impacts  on  the  environment  and  the  ecosystem  (Tacon  et  al.,  2011).   An   important   aspect   to   consider   when   using   alternative   protein   sources   for   fishmeal   is   to   utilize   sources  with  similar  amino  acid  profile.  Alternative  protein  sources  may  have  a  different  amino   acid  composition  than  the  desired  one,  and  thereby  causing  negative  effects  on  the  growth  of  the   fish.   It   is   therefore   of   importance   not   only   looking   at   the   crude   protein   level,   but   also   at   the   protein   composition   (Tacon,   1987).   The   amino   acids   can   be   divided   into   essential   and   non-­‐ essential  amino  acids.  The  essential  amino  acids  in  fish  feed  are:  arginine,  histidine,  isoleucine,   leucine,   lysine,   methionine,   phenylalanine,   threonine,   tryptophan,   and   valine.   Requirements   of   essential  amino  acids  for  fish  at  varying  dietary  protein  levels  are  displayed  in  Table  1  (Tacon,   1987).   Alriksson   et   al.,   (2014),   showed   that   P.   variotii   cultivated   on   spent   sulphite   liquor   permeate   consisted   of   similar   amounts   of   the   important   amino   acids   as   fishmeal   with   comparable  protein  content.   Table  1  –  Dietary  essential  amino  acid  (EAA)  requirements  (%  of  dry  diet)  of  fish  at  varying  dietary   protein  levels  (Tacon,  1987).  

Amino  acid  

Dietary  protein  level  (%)   45                                                                                    50                                                                                    55   1.94   2.15   2.37   0.31   0.35   0.38   0.82   0.91   1.00   1.26   1.40   1.54   2.30   2.55   2.81   2.66   2.96   3.25   0.87   0.96   1.06   1.31   1.45   1.60   1.04   1.15   1.27   1.45   1.61   1.77   0.27   0.30   0.33   1.50   1.66   1.83  

Arginine   Cystine*   Histidine   Isoleucine   Leucine   Lysine   Methionine   Phenylalanine   Tyrosine*   Threonine   Tryptophan   Valine     *Non-­‐essential  amino  acid.  

   

 

 

7  

Production  of  Single  Cell  Protein  from  Residual  Streams     from  2nd  Generation  Bioethanol  Production     2.1.1.1  Toxins  from  microorganisms   One  important  aspect  of  using  SCP  in  animal  feed  is  safety.  Some  microorganisms  can  produce   mycotoxins.   Mycotoxins   are   a   toxic   secondary   metabolite   expressed   by   the   kingdom   of   fungi.   Mycotoxins   are   associated   with   diseases   and   they   may   also   be   carcinogenic.   The   major   mycotoxins   include   aflatoxins,   deoxynivalenol,   fumonisins,   zearalenone,   T-­‐2   toxin,   ochratoxin,   and  certain  ergot  alkanoids  (Richard,  2007).   The  European  Food  and  Safety  Authority  have  set  up  guidance  values  for  the  maximum  content   of   mycotoxins   in   μg/kg   allowed   in   animal   feed   (Table   2)   (The   Commission   of   the   European   Communities,  2006)(The  Commission  of  the  European  Communities,  2003).  In  order  to  be  able   to  utilise  SCP  as  a  protein  source  in  fish  feed  the  content  of  mycotoxins  have  to  be  analysed.  In   the   study   performed   by   Alriksson   et   al.,   (2014),   the   concentrations   of   mycotoxin   in   P.   variotii   cultivated   on   spent   sulphite   liquor   permeate   were   well   below   the   recommended   maximum   values  given  by  the  European  Food  and  Safety  Authority.   Table   2   –   Directive   and   recommendation   from   the   European   Union   Commission   regarding   maximum  values  of  some  mycotoxins  in  products  animal  feed.  

Substance   Aflatoxin  B1   Deoxynivalenol   Zearalenone   Ochratoxin  A   Fumonisin  B1  +  B2  

Maximum  content  in  μg/kg   5-­‐20   900-­‐12  000   100-­‐3000   50-­‐250   5000-­‐60  000  

  2.1.1.2  β-­‐glucan,  an  immunostimulant  present  in  microorganisms   Microbial   protein   may   need   additional   properties,   than   just   constituting   a   protein   source,   in   order   to   compete   with   vegetable   protein   as   an   alternative   protein   source   in   fish   feed.     Microorganisms   can,   in   addition   to   protein   and   lipids,   also   contain   other   substances   that   can   improve  the  quality  of  the  fish  feed.   β-­‐glucans  are  a  group  of  molecules  generally  called  “biological  response  modifiers”  due  to  their   physiological  activeness.  β-­‐glucans  constitutes  a  structural  component  in  the  cell  walls  of  fungi,   yeast,   bacteria,   seaweed   and   some   plants   (Vetvicka   et   al.,   2013).   It   is   a   homopolysaccharide   consisting  of  glucose  molecules  linked  together  by  glycosidic  bonds  (Meena  et  al.,  2013).  The  cell   wall  further  consists  of  chitin,  other  hemicelluloses,  and  mannans  (Kyanko  et  al.,  2013).   β-­‐glucans   are   interesting   molecules   due   to   their   bioactive   and   medicinal   properties.   Some   of   them   are   anti-­‐microbial,   anti-­‐viral,   and   immune   stimulating   (Kyanko   et   al.,   2013).   Within   the   aquaculture   industry   the   risks   and   occurrence   of   diseases   and   infections   increases   along   with   the  increasing  production.  Due  to  the  concern  regarding  use  of  antibiotics  alternative  strategies   are   requested.   Alternative   approaches   can   include   use   of   vaccine,   dietary   supplement   of   probiotics,  prebiotics,  and  immunostimulants.  Immunostimulants  is  an  effective  tool  to  enhance   the   resistance   against   infectious   diseases   through   improvement   of   the   immune   system.   Immunostimulants  can  enhance  the  innate  humoral  and  cellular  defence  mechanism.  β-­‐glucans    

8  

Production  of  Single  Cell  Protein  from  Residual  Streams     from  2nd  Generation  Bioethanol  Production     have   shown   to   be   one   of   the   most   promising   immunostimulants   within   aquaculture   (Meena   et   al.,   2013).   Studies   with   β-­‐glucan   have   shown   to   affect   the   growth,   survival,   resistance   and   protection  against  pathogen,  antibody  production,  and  immune-­‐related  gene  expression  in  many   fish  species.  It  has  been  shown  that  the  dosages,  quality,  time  of  administration  and  duration  of   treatments   of   β-­‐glucan   is   of   great   importance   for   the   enhancement   of   various   parameters   related  to  growth,  survival,  and  immunity  (Meena  et  al.,  2013).     β-­‐glucans  is  today  routinely  used  in  commercial  aquaculture  production  (Vetvicka  et   al.,  2013).   The  potential  of  using  β-­‐glucan  as  prebiotics  (a  non-­‐digestible  feed  ingredient  which  stimulate   growth  and  activity  of  beneficial  bacteria  in  the  gastro  intestinal  tract)   is  an  interesting  aspect   for  future  investigation  (Meena  et  al.,  2013).     Kyanko  et  al.,  (2013)  studied  the  content  of  total  dietary  fibre,  and  especially  the  amount  of  β-­‐ glucan   in   thirty-­‐seven   different   filamentous   fungi,   including   P.   variotii.   P.   variotii   obtained   the   highest   amount   of   total   dietary   fibre   (51.7%)   and   the   highest   content   of   β-­‐glucan   (23.8%),   values   being   far   higher   than   earlier   reported   values   in   Basidiomycetes   and   yeast.   Today,   the   company   Biofeed   Technology   Inc.   produces   β-­‐glucan   by   production   and   derivation   from   specific   Paecilomyces   ssp.,   Saccharomyces   cerevisiae   and   Ganoderma   lucidum   (Biofeed   Technology   Inc.,   2014).  

2.2  Microorganisms  for  single  cell  protein  and  single  cell  oil  production   Various  bacteria,  yeasts,  fungi  and  algae  have  been  tested  and  investigated  for  production  of  SCP   and  SCO  throughout  the  years.  The  four  microorganisms  used  in  this  study  are  described  below.  

2.2.1  Paecilomyces  variotii   Paecilomyces   variotii   is   a   filamentous   fungus   belonging   to   the   order   Eurotiales   within   the   phylum  of  Ascomycota.  The  specie  is  commonly  found  in  soil,  wood,  and  food  (Houbraken  et  al.,   2010).   P.  variotii  has   a   history   of   being   used   for   SCP   production,   and   it   has   the   ability   to   grow   in   various  complex  residual  streams  from  different  industries  (Almeida  e  Silva  et   al.,  1995).  In  the   70’s,   the   Pekilo   process   was   started   in   Finland.   In   a   continuous   fermentation   process   the   filamentous  fungi  was  fed  with  spent  sulphite  liquor  from  a  pulp  mill  in  order  to  produce  SCP.   The   protein-­‐rich   fungus   was   even   approved   as   animal   feed   in   Finland   (Romantschuk,   1976),   although  the  process  is  currently  not  running  (Ugalde  and  Castrillo,  2002).  The  investigation  of   using  P.  variotii  for  SCP  production  has  continued,  even  though  the  interest  has  been  quite  low   the   last   decades.   Bajpai   and   Bajpai   (1987)   investigated   SCP   production   form   rayon   pulp   mill   waste,  Almeida  e  Silva  et   al.,  (1995)  investigated  using  eucalyptus  hemicellulose  hydrolysate  as   substrate   for   production   of   SCP.   In   a   recent   study   by   Alriksson   et   al.   (2014),   spent   sulphite   liquor  permeate  was  used  as  substrate  for  production  of  SCP.   P.  variotii  has  shown  to  consume  both  hexoses  (C6  sugars)  and  pentoses  (C5  sugars),  and  it  can   also   metabolise   aliphatic   acids   like   acetic   acid   and   formic   acid   (Alriksson   et   al.,   2014).   Cultivation  is  commonly  performed  at  30°C  and  at  pH  6.0  (Bajpai  and  Bajpai,  1987)(Almeida  e   Silva  et  al.,  1995)(Alriksson  et  al.,  2014).  

 

9  

Production  of  Single  Cell  Protein  from  Residual  Streams     from  2nd  Generation  Bioethanol  Production    

2.2.2  Cunninghamella  echinulata  and  Mortierella  isabellina   Cunninghamella   echinulata   and   Mortierella   isabellina   are   two   oleaginous   filamentous   fungi   belonging   to   the   order   of   Mucorales   within   the   phylum   Zygomycetes   (Fakas   et   al.,   2009)(Chatzifragkou   et   al.,   2010).   Strains   from   the   phylum   Zygomycota   are   considered   to   be   potential   producers   of   SCO   that   contain   γ-­‐linolenic   acid   (GLA)   (Chatzifragkou   et  al.,   2010).   It   has   been   reported   that   these   two   strains   show   variations   within   their   way   to   regulate   the   lipid   accumulation   process   when   cultivated   in   nitrogen-­‐limited   media.   It   has   also   been   shown   that   depending  on  what  type  of  sugars  that  are  metabolised  the  fatty  acid  composition  of  the  lipids   can   vary   (Chatzifragkou   et  al.,   2010).   The   genera   of   Cunninghamella  may   also   be   considered   as   a   relevant  microorganism  for  production  of  SCP  (Ugalde  and  Castrillo,  2002).  C.  echinulata  and  M.   isabellina  are  normally  cultivated  at  28°C  and  at  pH  6.0  (Fakas  et  al.,  2009)(Chatzifragkou  et  al.,   2010).   Zeng   et  al.   (2013)   showed   that   M.  isabellina   preferable   use   C6   sugars   compared   to   C5   sugars.   They   also   found   M.  isabellina   to   be   more   sensitive   to   degradation   products   from   lignin   than   to   other  inhibitory  compounds  commonly  formed  during  pretreatment  of  lignocellulose.  Aliphatic   acids   like   acetic   acid   and   formic   acids   even   improved   the   growth   and   lipid   production   at   low   concentration   (Zeng   et   al.,   2013).   Zeng   et   al.,   also   showed   that   up   to   12.6   g/L   of   mycelium   containing   34%   lipids   could   be   obtained   when   cultivating   M.   isabellina   on   wheat   straw   hydrolysate  (pretreated  with  dilute  sulphuric  acid)  for  6  days.  

2.2.3  Yarrowia  lipolytica   The   oleaginous   yeast   strain   Yarrowia   lipolytica   is   strictly   aerobic   and   it   produces   many   important   metabolites   and   has   a   high   secretory   activity.   It   is   considered   to   be   non-­‐pathogenic   and  the  Food  and  Drug  Administration  (FDA,  USA)  has  classified  many  processes  based  on  this   microorganisms   as   generally   regarded   as   safe   (GRAS)   (Coelho   et  al.,   2010).   Y.  lipolytica   can   be   described   as   an   industrial   workhorse,   being   used   for   a   broad   range   of   applications.   Some   products   that   can   be   produced   using   Y.  lipolytica   are   for   example   lipase   and   different   organic   acids   (e.g.   citric   acid)   (Coelho   et   al.,   2010).   Considering   SCP   and   SCO,   Y.   lipolytica   is   mainly   considered   as   an   SCO   producer   but   can   also   be   used   for   SCP   production   (Ugalde   and   Castrillo,   2002).  Cultivation  is  usually  performed  at  a  temperature  between  28°C  and  30°C  and  at  pH  5.5-­‐ 6.0  (Makri  et  al.,  2010)(Katre  et  al.,  2012).   Y.  lipolytica   can   utilize   several   different   hexoses   as   carbon   source,   for   example   glucose,   fructose,   and  mannose.  The  yeast  can  also  degrade  acids  like  acetic,  lactic,  propionic,  malic,  succinic,  citric,   and   oleic   acid   and   use   those   as   the   sole   carbon   and   energy   source.   Additionally,   ethanol   and   glycerol  can  also  be  used  as  carbon  source  (Coelho  et  al.,  2010).  Ethanol  concentrations  up  to  3%   can   be   used   as   carbon   source   before   it   becomes   too   toxic   (Barth   and   Gaillardin,   1997).   Other   carbon   sources   that   can   be   utilized   are   n-­‐alkenes   and   1-­‐alkenes   (Barth   and   Gaillardin,   1997).   Tsigie   et  al.,   (2011),   showed   that   some   strains   of   Y.  lipolytica   can   utilize   pentoses   as   a   carbon   source.   They   also   showed   that   biomass   formation   could   be   inhibited   by   degradation   products   commonly  found  in  lignocellulose  hydrolysates,  such  as  HMF  and  furfural  (Tsigie  et  al.,  2011).      

 

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Production  of  Single  Cell  Protein  from  Residual  Streams     from  2nd  Generation  Bioethanol  Production    

2.3  Bioethanol  production  from  lignocellulose   Production   of   ethanol   from   lignocellulose   is   usually   performed   through   pretreatment   and   hydrolysis   of   the   lignocellulosic   material,   followed   by   fermentation   and   distillation.   Lignocellulosic   biomass   is   an   interesting   feedstock   due   to   its   abundance   and   its   lower   cost   compared   to   other   substrates,   such   as   sugar   and   starch   from   agricultural   crops   (Ruan   et   al.,   2012).   In   addition,   lignocellulosic   biomass   has   fewer   competing   uses,   compared   to   crops   or   grains,  and  does  not  compete  with  food  supply  (Huang  et  al.,  2013).  

2.3.1  Lignocellulose   Lignocellulose  consists  mainly  of  cellulose,  hemicellulose,  lignin,  extractives,  and  ash  (Figure  2).   Depending  on  the  origin  of  the  lignocellulose  both  the  chemical  and  the  structural  composition   varies   (Table   3).   Examples   of   lignocellulose   sources   are   grasses,   hardwood,   softwood,   and   agricultural  residues  such  as  wheat  straw  and  sugarcane  bagasse  (Balat,  2011).     Cellulose  is  a  linear  homopolysaccharide  consisting  of  β-­‐D-­‐glucopyranose  units  linked  together   by   1,4-­‐β-­‐glycosidic   bonds.   Parallel   cellulose   polymers   are   held   together   via   hydrogen   bonds   and   form  microfibrils,  which  in  turn  form  fibrils  that  build  up  the  cellulose  fibres  (Sjöström,  1993)   (Berg   et   al.,   2007).   The   fibres   support   the   plant   cell   wall   and   give   it   its   strength   and   rigidity.   Cellulose   is   the   major   constituent   in   plant   biomass   and   make   up   around   30-­‐45%   of   the   dry   weight  (Balat,  2011).     Hemicellulose,  around  20-­‐40%  of  the  dry  weight  of  lignocellulose,  is  a  short  and  highly  branched   heteropolysaccharide   consisting   of   both   pentoses   (five-­‐carbon   sugar)   and   hexoses   (six-­‐carbon   sugar).  The  most  dominant  monosaccharides  in  hemicellulose  are  xylose,  arabinose,  which  are   pentoses,   and   galactose,   glucose   and   mannose,   which   are   hexoses   (Balat,   2011).   Xylose   is   the   most   abundant   sugar   in   the   hemicellulose   of   agricultural   residues   while   mannose   is   the   most   abundant  sugar  in  the  hemicellulose  of  softwoods  (Balat,  2011).     Lignocellulose   consists   of   approximately   15-­‐30%   lignin   (Balat,   2011).   Lignin   is   an   aromatic   polymer   consisting   of   phenylpropane   units   where   the   residues   are   linked   together   to   a   very   complex   structure.   Precursors   to   lignin   are   p-­‐coumaryl   alcohol,   coniferyl   alcohol,   and   sinapyl   alcohol  (Sjöström,  1993).     Extractives   are   non-­‐structural   components   and   include   e.g.   phenols,   tannins,   fats,   and   sterols   (Martínez   et   al.,   2005).   Non-­‐extractives   present   in   lignocellulose   mainly   consist   of   ash   components,  such  as  silica  and  alkali  salts.  The  amount  of  ash  in  straw  can  be  as  high  as  up  to   10%   while   the   amounts   are   commonly   very   low   in   wood   materials,   below   1%   (Klinke   et   al.,   2004).    

 

11  

Production  of  Single  Cell  Protein  from  Residual  Streams     from  2nd  Generation  Bioethanol  Production     Table  3  –  Composition  for  different  types  of  lignocellulosic  materials  (%  dry  weight)  (Balat,  2011).  

Material   Cellulose   Grasses   25-­‐40   Hardwoods   45  ±  2   Softwoods   42  ±  2   Wheat  straw   37-­‐41  

Hemicelluloses   Lignin   25-­‐50   10-­‐30   30  ±  5   20  ±  4   27  ±  2   28  ±  3   27-­‐32   13-­‐15  

Extractives   -­‐   5  ±  3   3  ±  2   7  ±  2  

Ash   -­‐   0.6  ±  0.2   0.5  ±  0.1   11-­‐14  

2.3.2  Hydrolysis  and  pretreatment     Hydrolysis   of   lignocellulose   can   be   performed   chemically   or   biologically   (i.e.   enzymatic   hydrolysis).   In   the   hydrolysis,   hemicellulose   and   cellulose   are   degraded   into   monosaccharides.   The   two   main   methods   for   chemical   hydrolysis   are   concentrated-­‐acid   hydrolysis   and   dilute-­‐acid   hydrolysis   (Galbe   and   Zacchi,   2002).   Both   the  concentrated-­‐   and  dilute-­‐acid  hydrolysis   methods   use   acid   as   catalyst   (e.g.   H2SO4,   SO2)   along   with   high   temperatures.   In   dilute-­‐acid   hydrolysis   acid   concentrations   below   4%   and   temperatures   ranging   from   140-­‐200°C   are   commonly   used.   The   acid   works   as   a   catalyst   by   breaking   the   up   the   structure   of   the   cellulose,   hemicelluloses,   and   lignin,   thereby   making   the   hydrolysis   with   water   easier.   Chemical   hydrolysis   can   also   be   performed  using  an  alkali  catalyst  (e.g.  NaOH,  NH3,  Ca(OH)2)  (Galbe  and  Zacchi,  2012).   Hydrolysis   of   the   lignocellulose   can   also   be   performed   enzymatically.   In   the   process   of   enzymatic   hydrolysis   a   mixture   of   different   enzymes,   consisting   mainly   of   cellulases   and   hemicellulases,   is   used   (Galbe   and   Zacchi,   2012).   To   enable   efficient   enzymatic   hydrolysis   of  the   lignocellulose   a   pretreatment   step   is   typically   required.   The   aim   with   the   pretreatment   is   to   open   the   structure   of   the   lignocellulose,   and   make   the   hemicellulose   and   cellulose   more   accessible   for   degradation   into   monosaccharides   (Almeida   et   al.,   2007).   Pretreatment   can   be   divided   into   four   categories:   physical,   chemical,   physio-­‐chemical,   and   biological.   The   thermochemical   method   “steam   explosion”   is   a   commonly   used   method.   Steam   explosion   is   typically  performed  at  temperatures  between  160-­‐240°C  for  a  short  time,  1-­‐20  minutes  (Galbe   and  Zacchi,  2012).  The  residual  streams  used  in  this  project  were  collected  from  a  process  based   on  steam  explosion  with  acid  catalyst.     Depending  on  the  type  of  pretreatment  and  hydrolysis  method,  different  types  and  amounts  of   degradation  products  are  generated  (Figure  2).  Some  of  these  products  can  act  as  fermentation   inhibitors.   The   severity   of   the   pretreatment   effects   the   formation   of   degradation   products   (Klinke  et  al.,  2004).  

 

12  

Production  of  Single  Cell  Protein  from  Residual  Streams     from  2nd  Generation  Bioethanol  Production     Macromolecules+

Cellulose'30*40%'

Sugar+ components+

Fermenta2on+ inhibitors+

Glucose' Mannose' Galactose'

Xylose' Arabinose' Hemicellulose' 20*30%' Uronic'acid' Ace:c'acid'

Lignin'20*30%'

Extrac:ves'

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