Evaluation  of  Cellruptor  pre-treatment  on                            biogas  yield  from  various  substrates    

 

 

 

SELVAKUMAR  THIRUVENKADAM  

 

 

 

 

 

 

 

Supervisor:    Mr  Andreas  Berg                                              Research  Manager                                              SCANDINAVIAN  BIOGAS  FUELS  AB                                              SE-581  83  Linköping  SWEDEN  

 

Examiner:      Prof.  Gen  Larsson                                            Head  of  Div.  Bioprocess  Technology                                            Department  of  Biotechnology                                            KTH  ROYAL  INSTITUTE  OF  TECHNOLOGY                                            SE-106  91  Stockholm  SWEDEN  

 

I  

 

ABSTRACT  

In  this  thesis  work,  Cellruptor  pre-treatment  was  evaluated  in  order  to  increase  biogas  yield.  Initially,  the  effects  of  residence  time  (30,  60,  90,  120  and  180  min)  and  substrate  release   (rapid/non-rapid)   from   the   draining   port   of   Cellruptor   on   biosludges   were  investigated   to   find   the   optimum   operating   conditions   of   Cellruptor.   Under   these  optimum  operating  conditions,  the  effect  of  Cellruptor  pre-treatment  on  batch  reactors  of   various   substrates   and   semi-continuous   digester   of   biosludge   were   investigated   at  mesophilbiosludge,  dewatered  sludge,  digested  sludge,   fibre  sludge,  hay,  maize  silage,  minced  meat,  orange  peel,  seaweed  and  yeast.  From  the  initial  study,  90  min  residence  time  and  rapid   release   of   pre-treated   substrate   from   draining   port   were   found   to   be   optimum  operating   conditions   of   Cellruptor.   From   the   batch   experiments,   Cellruptor   pre-treatment  showed  maximum  and  minimum  increase  of  methane  yield  in  hay  (32%)  and  dewatered  sludge  (2%)  respectively.  The  semi-continuous  digester  experimental  results  showed   increase   in   biogas   production   by   22.4%   from   Cellruptor   pre-treatment   of  biosludge   at   HRT   of   15   days   and   OLR   of   2.0   g   VS/L/day.   With   further   studies,  Cellruptor   pre-treatment   may   be   deployed   in   large-scale   biogas   plants   to   improve  biogas  yield.  

 

Keywords:    Cellruptor,  pre-treatment,  biogas,  methane,  biosludge,  mesophilic,  batch    

   

II  

 

 Contents  

ABSTRACT   I  

CONTENTS   II  

1   INTRODUCTION   1  

1.1   Aim   2  

1.2   Hypothesis   2  

1.3   Strategy   2  

2   BACKGROUND   3  

2.1   Biogas    for  a  sustainable  environment   3  

2.2   Anaerobic  Digestion   3  2.2.1   Microbiology  and  Biochemistry   4  2.2.2   Environmental  factors   5  2.2.3   Solid  Characteristics   6  2.2.4   Operational  Parameters   6  2.2.5   Control  Parameters   7  

2.3   Pre-treatment  Techniques  of  substrates   7  2.3.1   Cellruptor   9  

2.4   The  substrates   9  2.4.1   Biosludge   9  2.4.2   Dewatered  Sludge   9  2.4.3   Digested  Sludge   10  2.4.4   Fibre  Sludge   10  2.4.5   Hay   10  2.4.6   Maize  Silage   10  2.4.7   Minced  Meat   10  2.4.8   Orange  Peel   11  2.4.9   Seaweed   11  2.4.10   Yeast   11  

3   METHODS  AND  MATERIALS   12  

3.1   Cellruptor   12  3.1.1   Process  conditions   12  

3.2   Batch  Experiment   13  3.2.1   The  substrates  &  Experimental  set-up   13  

III  

 

3.2.2   Batch  start  up   13  3.2.3   Analysis   16  

3.3   Semi-continuous  digester  experiment   17  3.3.1   The  substrates   17  3.3.2   Digester  configuration   17  3.3.3   Digester  Operation   17  3.3.4   Analysis   18  

3.4   Software   18  

4   RESULTS   20  

4.1   Batch  experiments   20  4.1.1   Batch  set  A   20  4.1.2   Batch  set  B   21  4.1.3   Batch  set  C,  D  and  E   22  

4.2   Semi-continuous  digester  experiment   26  

5   .      DISCUSSION   28  

5.1   Batch  experiments   28  5.1.1   Biosludge   28  5.1.2   Dewatered  Sludge   29  5.1.3   Digested  Sludge   29  5.1.4   Fibre  sludge   29  5.1.5   Hay   30  5.1.6   Maize  Silage   30  5.1.7   Minced  Meat   30  5.1.8   Co-digestion  of  minced  meat  with  digested  sludge   31  5.1.9   Orange  Peel   31  5.1.10   Seaweed   31  5.1.11   Yeast   32  

5.2   Semi-continuous  digester  experiment   32  5.2.1   Biogas  production  and  Methane  content   32  5.2.2   pH,  VFA  and  VS  reduction   33  

5.3   Evaluation  of  Cellruptor  Pre-treatment   33  

6   .  CONCLUSION   35  

7   REFERENCES   36  

ACKNOWLEDGEMENT   42          

IV  

 

APPENDIX  A:  Statistics  Sweden   43  APPENDIX  B:  Batch  raw  data   44  APPENDIX  C:  Statistical  data   47  

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1 INTRODUCTION    

For   the   past   few   decades,   the   increasing   global   scarcity   of   petroleum   and   petroleum-derived  fuels  has  led  to  intensive  research  on  finding  new  alternative  energy  sources  for  power   generation   and   transportation   all   over   the   world.   Among   the   proposed  alternative  fuels,  biogas  has  received  much  attention  in  recent  years  for  gas  engines  and  could   be   one   remedy   in   many   countries   to   reduce   their   oil   imports.   The   European  Union   (EU)   renewable   energy   policy   has   set   a   target   to   produce   renewable   energy,  which  meets  20%  of  European  energy  demand,  by  2020,  while  biogas  contributes  25%  share   of   this   renewable   energy   (Nielsen   and   Oleskowicz-Popiel).   Among   the   EU  nations,   Germany   remains   top   in   biogas   production,   where   major   amount   of   biogas  (85%)   is   produced   from   municipal   solid   waste   methanisation   plant,   decentralised  agricultural  plant  and  centralised  co-digestion  plant   .  

 

In   Sweden,   biogas   is   been   produced   since   1940   from   sewage   treatment   plants.   The  biogas  production  from  other  organic  

substrates.  Biogas  production  from  sugar  refinery  plants  and  paper  mills  were  initiated  during   this   period.   Every   Swedish   municipality   constructed   biogas   plants   at   their  sewage   treatment   facility   to   enhance   extraction   of  methane  gas  from  landfills  were  innovated  to  minimise  these  methane  emission  to  the  atmosphere.   Large   scale   anaerobic   co-digestion   of   various   organic   substrates   such   as  agricultural   waste,   food   waste,   slaughterhouse   waste,   etc.,   was   developed   during  

  This   led   to   continuous   research   and   development   in   the   field   of   biogas  technology.    

 

With  the  aim  of  becoming  w -free  economy  by  2020,  Swedish  government  has   implemented   new   renewable   energy   policies   to   promote   the   renewable   energy  production   in   Sweden   (Swedish   energy   agency,   2011;   EREC,   2011).   Being   a   leader   of  biogas-to-vehicle-fuel   revolution,   amount   of   biogas   delivered   as   a   vehicle   fuel   is  substantially   higher   than   natural   gas   supplies   in   Sweden   (SCB,   2011;   Appendix   A).  According  to  Avfall  Sverige  (Swedish  Waste  Management),  317,440  MWh  of  biogas  was  produced  by  anaerobic  digestion  of  green  and  food  waste  in  2009,  which  is  equivalent  to  35  million  litres  of  petrol.  Table  1  represents  the  primary  biogas  production  in  2009  from  Germany  (leading  biogas  producer  in  EU),  Sweden  and  EU  (EurO  

 

Considering   perspectives   of   bioenergy   systems   and   waste   management,   the   biogas  production   from   various   waste   materials   has   been   gaining   more   attention   in   the   last  couple   of   years.   Further   increase   in   biogas   production   can   be   accomplished   by  improving   the   biodegradability   on   pre-treating   the   substrates.   An   ample   scope   on  

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research   and   applications   of   anaerobic   digestion   and   various   pre-treatment   strategies  has   been  made   in   this   thesis   with   emphasis   on   combining   both   topics   to   enhance   the  biogas  production  from  various  waste  materials.  

 

Table  1:  Biogas  production  in  Germany,  Sweden  and  EU,  in  2009    

Region  Primary  biogas  output  (ktoe)  

Landfills   Sewage  sludge*   Others**   Total  

Germany      265.5  (6%)      386.7  (9%)   3561.2  (85%)   4213.4  

Sweden          34.5  (31%)          60.0  (55%)          14.7  (14%)      109.2  

EU   3001.6  (36%)   1003.7  (12%)   4340.7  (52%)   8346.0  

*  Urban  and  Industrial  sludge      **  Municipal  solid  waste  methanisation  plant,  decentralised  agricultural  plant  and    centralised  co-digestion  plant  

 

1.1 Aim  This   thesis  work  evaluates   the  Cellruptor  pre-treatment   to  enhance  biogas  production  from  various  substrates.  

 

1.2 Hypothesis  Cellruptor  pre-treatment  will  improve  anaerobic  digestion  process.  

 

1.3 Strategy   Optimum  Cellruptor  operating  conditions  were  analysed  after   investigating  the  

effect   of   cellruptor   residence   time   variation   and   rapid/non-rapid   release,   on  methane  yield  of  biosludge.  

Batch   experiments   were   performed   to   evaluate   the   cellruptor   pre-treatment   on  biogas  production  from  various  substrates,  namely:  biosludge,  digested  sludge,  dewatered   sludge,   fibre,   hay,   maize,   minced   meat,   orange   peel,   seaweed   and  yeast.  

A   semi-continuous   digester   experiment   was   also   carried   out   to   study   the   pre-treatment  effect  on  methane  yield  from  biosludge.  

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2 Background  2.1 Biogas    for  a  sustainable  environment  Biogas   is  a  renewable  energy  source,  comprising  of  methane  (50-80%),  carbon  dioxide  (20-50%),  and  traces  of  other  gases  such  as  hydrogen,  carbon  monoxide,  and  nitrogen.  In   large   scale,   biogas   can   be   used   for   production   of   heat   and/or   steam,   electricity,  chemicals   and   fuel   cells   whereas   in   small   scale,   it   remains   as   an   alternative   energy  source  in  rural  communities,  which  meets  the  basic  need  of  cooking  and  lighting.  Once  upgrading  biogas  to  high  purity  level  adequate  to  vehicle  fuel  standards,  it  can  be  used  as   vehicle   fuel   similar   to   natural   gas.   Biogas   can   be   produced   by   many   ways   which  includes   pyrolysis,   hydrogasification   and   anaerobic   digestion,   while   anaerobic  digestion   remains   as   a   most   promising   technology   for   developing   a   sustainable  environment.   At   the   environmental   level,   biogas   production   forbids   the   release   of  greenhouse  gas  (methane)  into  the  atmosphere  and  also  replaces  the  chemical  fertilizers  with  nutrient  rich  digestate.  (Engler  et  al.,  1998)  

 

2.2 Anaerobic  Digestion  Anaerobic   digestion   is   a   biological   process   which   is   capable   of   converting   almost   all  types  of  organic  materials   into  methane  and  carbon  dioxide.  Some   existing  sources  of  methane  emissions  are  wetland  soils,  oceans,  rumen  of  ruminant  animals,  and  the  lower  intestinal   tracts   of   humans,   landfills,   and   sewage   digesters.   Microbial   production   of  methane  from  organic  matter  has  become  an  attractive  method  of  waste  treatment  and  resource   recovery,   and   this   is   carried   out   by   action   of   complex   anaerobic   flora  consisting   of   bacteria,   fungi,   protozoa   and   archaeal   methanogens.   Anaerobic   process  also  offers  an  effective  means  of  pollution  reduction,  which  is  superior  to  that  achieved  via   conventional   aerobic   process   due   to   the   fugitive   volatile   emissions   taking   place  before   degradation   in   aerobic   treatment   plants   leading   to   air   pollution.   Methane  produced  by  anaerobic  fermentation  of  biomass  is  a  clean,  renewable  fuel.    

Three  basic  points  about  anaerobic  digestion  process  are:  

(i)  Slow  growing  anaerobic  bacteria  and  archaeal  methanogens  are  the  most   important  microbial  community  involved  in  biogas  production  process;  

(ii)  A  higher  level  of  metabolic  specialization  could  be  seen  in  this  process  than  aerobic  process;  

(iii)  Most  of  the  substrate  free  energy  is  converted  to  terminal  product  methane.  At  the  end   of   digestion,   the   end   product   contains   less   microbial   biomass   than   aerobic  decomposition  and,  therefore,  disposal  of  digested  sludge  after  digestion  may  not  be  a  problem   but   it   also   depends   on   the   feedstock   characteristics.   (Nagamani   and  Ramasamy,  1999)                        

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As   practiced   for   several   years,   interest   in   anaerobic   digestion   in   many   countries   has  widely   focused   on   the   economic   recovery   of   fuel   gas   from   municipal   sludge,   cattle,  industrial  and  kitchen  wastes  and  agricultural  surpluses  (Demirbas  et  al.,  2011).    

 

2.2.1 Microbiology  and  Biochemistry  Hydrolysis,  acidogenesis,  acetogenesis  and  methanogenesis  are  four  important  steps  of  anaerobic  digestion  process.  The  model  of  microbial  groups   involved  in   this   four-step  flow   of   carbon   from   complex   polymers   to   biogas   consists   of   five   groups.   During   the  process   of   anaerobic   digestion   (Figure   1),   complex   polymers   are   broken   into   simple  products  by  enzymes  produced  by  fermentative  bacteria  (Group  1),  which  ferment  the  substrate   to   short   chain   fatty   acids,   hydrogen   and   carbon   dioxide.   Fatty   acids,   longer  than   acetate   are   catabolized   to   acetate   by   obligate   hydrogen   producing   acetogens  (Group  2).  Hydrogen,  carbon  dioxide  and  acetate  are  the  major  products  produced  by  these  two  groups  after  digestion  of  the  substrate.  Hydrogen  and  carbon  dioxide  can  be  converted   into   acetate   by   hydrogen   oxidizing   acetogens   (Group   3)   or   methane   by  carbon   dioxide   reducing,   hydrogen   oxidizing   methanogens   (Group   4).   Acetate   is   also  converted  into  methane  by  acetotropic  methanogens  (Group  5)  (Show  et  al.,  2010).    

 Figure  1:  Steps  in  Anaerobic  Digestion    

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2.2.2 Environmental  factors    

Nutrients  

Carbon,   nitrogen   and   phosphorus   are   the   macro   nutrients   that   nourish   the   microbial  growth.   Generally,   these   nutrients   are   available   in   sufficient   quantities   in   municipal  sewage   and   sludge.   Microbial   community   also   relies   on   micro   nutrients   such   as  sulphur,  vitamin  and  trace  of  minerals  (iron,  cobalt,  nickel,  molybdenum,  selenium).  All  nutrients  should  be  available   in  sufficient  quantities  as   the  microbial  activity  depends  on   the  multiplicative   factor  of   all   essential   nutrients.   There   should   also  be  a  balanced  proportion  of  carbon  and  nitrogen  and  the  optimum  proportion  ranges  between  20:1  to  30:1  (C:N  ratio)  (Davidsson,  2007).    

Temperature  

Digestion   temperature   remains   a   crucial   factor   in   anaerobic   processes   and   it   has   to  remain   constant   throughout   the   process.   The   operational   temperature   ranges   of  anaerobic   digestion   process   are   classified   as   mesophilic   (30- -

-used  as  the  thermophilic  process  has  disadvantages  such  as  process  instability,  lowered  effluent   quality,   low   methane   production   per   unit   substrate   and   high   energy  requirement  for  heating,  and  maintenance  (Duran  and  Speece,  1997,  Vindis  et  al.,  2009).  Psychrophilic  conditions  are  seldom  used  due  to  the  slow  microbial  growth.  

pH  

The  enzymatic  activity  of  methanogenic  bacteria  is  regulated  within  a  specific  pH  range  and  the  maximum  activity  is  achieved  at  optimum  pH.  In  mesophilic  anaerobic  process,  the  desired  pH  range  for  methanogens  are  6.6-7.6  (optimum  pH  around  7.0)  and  6.6-7.8  (optimum  pH  6.8)  under  low  solids  (1-2%)  and  high  solids  (90-96%)  sludge  respectively  (Lay  et  al.,  1997).  The  inhibition  of  methane  formation  might  also  occur,  when  the  pH  is  lesser  than  6.3  or  higher  than  7.8  during  digestion  of  high  solids  sludge  (Liu  et  al.,  2008).  

Alkalinity  

The   accumulation   of   volatile   fatty   acids   (VFA)   and   the   production   of   carbon   dioxide  during  anaerobic  digestion  can  result  in  a  pH  drop,  which  may  cause  process  instability  and   inhibition   of   methanogenesis.   Thus,   the   addition   of   external   alkalinity   source  (buffering   agents)   leads   to   achieve   stable   pH   and   may   improve   the   rate   of   anaerobic  digestion  (Couderc  et  al.,  2008).  The  bicarbonate  of  the  liquid  phase  and  carbon  dioxide  in  gas  phase  stabilizes  the  system  pH  by  producing  alkalinity,  which  counteracts  the  pH  reduction  by  accumulation  of  VFAs  (Appels  et  al.,  2008).  

Moisture  Content  

Water   is   important   as   the   nutrients   get   dissolved   in   it,   which   in   turn   facilitate   the  diffusion   transport   of   these   dissolved   substances   across   the   bacterial   cell   membrane.  Thus  addition  of  water  increases  the  rate  of  hydrolysis,  whereby  decreasing  the  rate  of  

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solids   accumulation   (Couderc   et   al.,   2008).   Lay,   et   al.   (1997)   investigated   the   effect   of  moisture  content  of  digesting  sludge  on  biogas  production.  

Toxic  Substances  

Volatile  fatty  acids  (VFAs),  free  ammonia,  hydrogen,  hydrogen  sulphide,  heavy  metals,  chlorinated   compounds   and   detergents   are   few   toxic   substances   that   inhibit   the  anaerobic  process.  These  substances  are  either  produced  during  the  digestion  process  or  already  present  in  the  substrates  and  hence,  few  substrates  need  to  be  pre-treated  before  AD,  to  remove  toxic  substances  (Show  et  al.,  2010).  

 

2.2.3 Solid  Characteristics    

Total  Solids  

Total  solids  (TS)  are  the  amount  of  dry  matter  remaining  after  the  removal  of  moisture  

TS   %   of   the   substrate   fed   into   the   reactor   has   no   effect   either   in   TS   or   VS   removal  (Fongsatitkul  et  al.,  2010).  

Volatile  Solids  

Volatile  solids  (VS)  are  the  amount  of  organic  matter   lost  on  combusting  dry  solids  at  

amount  of  organic  matter  present  in  the  waste.    

 

2.2.4 Operational  Parameters    

Organic  Loading  Rate  

Organic  loading  rate  (OLR)  is  the  measure  of  organic  material  fed  into  the  digester  and  this  depends  on  volatile  solids  content  and  methane  potential  of  the  substrate.  Feeding  the   digester   above   optimum   OLR   may   lead   to   accumulation   of   inhibitory   substances,  disturbing  the  process  stability  or  low  VS-reduction.  

Hydraulic  Retention  Time  

Hydraulic   retention   time   (HRT)   is   the   average   residence   time   of   the   liquid   inside   the  digester  and  the  optimum  HRT  for  most  mesophilic  anaerobic  digester  ranges  between  15  to  30  days  (Davidsson,  2007).      

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Temperature      (Refer  2.2.2)  

Stirring  

The   constituents   in   the   reactor   must   be   mixed   well   to   increase   contact   between   the  substrate   and   microorganisms.   It   provides   a   uniform   sludge   concentration   across   the  

 substrates.   Optimum   mixing   should   be   maintained   to   avoid   the   disruption   of  microorganism.   Different   substrates   in   co-digestion   process   should   be   mixed   well  before  entering  the  AD  process.  

 

2.2.5 Control  Parameters    

Volatile  Fatty  Acids  

Volatile   fatty   acids   (VFA)   are   intermediates   formed   during   the   digestion   process   and  when  VFAs  get  accumulated  in  high  quantities,  they  inhibit  Methanogenesis.  The  most  prominent   inhibitory  VFAs  are  acetic  and  propionic  acid.  At   increasing   temperatures,  accumulation  of  VFA  decreases  the  pH  value  and  when  pH  falls  below  6.0,  AD  process  gets  inhibited  (Nielsen  and  Angelidaki,  2008).  

Volatile  Solids  Reduction    

Volatile  solids  constitute  the  organic  portion  of  total  solids  and  these  reduce  during  the  digestion  process,  as  they  are  converted  to  biogas.    Volatile  solids  reduction  is  directly  related  to  the  biogas  yield  (Appels  et  al.,  2008).  

Methane  Potential  

Based  on  economical  aspect  of  AD,  it  is  important  to  know  the  methane  potential  of  the  substrates.   Many   techniques   such   as   biochemical   methane   potential   (BMP),   dynamic  respiration   rate   (DR4)   and   chemical   oxygen   demand   (COD)   test   are   available   to  determine   the  methane  yield.  The  most   common  BMP   test   is  a  batch   test   for  28  days,  which  is   likely  to  provide  information  useful  for  execution  of  CSTR  (Shanmugam  and  Horan,  2009).  

 

2.3 Pre-treatment  Techniques  of  substrates  The  digestion  process  is  affected  by  the  non-degradable  constituents  and  rigid  cell  wall  of   the   substrate   which   cause   the   cell   constituents   inaccessible   for   the   anaerobic  microorganisms   and,   hence,   the   anaerobic   digestion   is   limited   by   hydrolysis   rate  (Rivard   et   al.,   1998).   The   microbial   consortia   in   the   reactor   tend   to   multiply   by  metabolizing  the  organic  matter  and  forms  biomass.  The  reduction  of  substrate  biomass  is   an   important   factor   to   enhance   biogas   production   and   this   can   be   achieved   by   cell  lysis.   Hence,   an   effective   pre-treatment   aims   to   enhance   the   biogas   production   by  

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improving   the   substrate   accessibility   to   the   microbial   community   and   consequently,  accelerating  the  rate  of  anaerobic  digestion.    

During   recent   years,   many   studies   have   been   made   on   various   mechanical   pre-treatment  techniques  that  disrupt  cells  by  application  by  force,  including:  

 

High  pressure  homogenizer          

The  High  pressure  pump  compresses  the  sludge  up  to  several  bars  (up  to  900  bar)  and  then,   the   sludge   undergoes   a   sudden   depressurization   in   the   homogenizing   valve  forming   cavitation   bubbles.   An   irreversible   disruption   of   the   cell   membrane   happens  during  the  explosion  of  these  bubbles  (Rai  and  Rao,  2009).    

Ultrasonic  homogenizer                    

Cavities  or  microbubbles  are  formed  due  to  the  repetitive  compression  and  rarefaction  of   the   ultrasonic   waves,   when   passed   through   sludge   medium.   The   cell   wall   and  membranes  are  disrupted  due  to  the  powerful  mechanical  shear  force  generated  during  the  collapse  of  many  microbubbles  (Khanal  et  al.,  2007).  This  principle  is  an  adaptation  from  Pulsed  electric   field   technology,  which  has  notable  significances   in  medical   field  (imaging  device),  food  industry  (extraction  of  vegetable  oils),  etc.  

Thermal  Hydrolysis  

Cell  rupture  is  achieved  by  effect  of  heat  produced  at  high  temperature  (160-30-60   min)   leading   to   increase   in   sludge   digestion   and   soluble   COD   (Carrere   et   al.,  2008).  

Freezing  and  Thawing  

Freeze/thaw   pre-treatment   disrupts   the   cell   membrane   physically   by   forming   ice  crystals.   They   cause   irreversible   rupture   of   cell   floc   by   reducing   the   bound   water  content  (Gao,  2010).  

Gamma-irradiation  

Gamma   radiations   disrupt   the   cell   membrane   and   release   the   soluble   organic  compounds,   which   influences   the   hydrolysis   step   in   the   digestion   process   (Lafitte-Trouque  and  Forster,  2002).  

 

Besides   the   above   mentioned   mechanical   pre-treatments,   viz.   chemical   pre-treatment  (Acid  or  alkaline  hydrolysis,  Ozone  pre-treatment)  (Perez-Elvira  et  al.,  2006),  Biological  pre-treatments   (Yunqin   et   al.,   2010)   and   combination   of   pre-treatments   such   as  Microsludge®  (combination  of  chemical  and  mechanical  pre-treatment)  are  available  to  increase   the   digestion   rate.   Even   though,   there   is   existence   of   pre-treatments   in  commercial  level  such  as  Microsludge®,   ®hydrolysis  process  and  Crown®  

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pre-treatment   based   on   economical   and   operational   grounds.   One   such   promising  technology  is  Cellruptor.    

 

2.3.1 Cellruptor  Eco-Solids   International   Ltd.   (Hampshire,   UK)   has   developed   this   simple   cell  disintegration  technology,  Cellruptor  and,  reported  28%  increase  in  biogas  production  during   the   commercial   trial   period   at   Yorkshire   wastewater   treatment   plant   (WWTP)  (Yorkshire,   UK).   Unlike   other   pre-treatment   techniques,   Cellruptor   just   require   low  energy  (maximum  10  bar  pressure)  to  disrupt  the  cells.  

Principle    

A   soluble   gas   such   as   CO2   is   compressed   to   the   sludge   at   10   bar   pressure   and   this  soluble  gas  diffuses   to   the  cell   through  the  cell  wall.  During  a  rapid  depressurization,  the  diffused  CO2  causes  cell  expansion  leading  to  an  irreversible  rupture  of  cell  wall.  At  large   scale,   biogas   containing   40%   CO2   can   be   passed   to   the   Cellruptor   making   the  process  very  more  economical  than  spending  for  a  compressed  gas  tank.    

 

2.4 The  substrates    

2.4.1 Biosludge  Biosludge   is   the  outcome  of   the   secondary   (biological)   treatment   of   sewage   treatment  plants.  It  is  also  called  as  excess  sludge,  activated  sludge,  waste  activated  sludge  (WAS)  or  surplus  activated  sludge  (SAS).  Resulted  due  to  overproduction  of  microorganisms,  biosludge   contain   rich   biomass,   extracellular   polymeric   substances   (EPS)   with   more  than  95%  water   (Yin  et  al.,   2004).  The  biomass  comprises  of   Bacteria,   fungi,  protozoa,  and  rotifers.  Generally,  the  TS  and  VS  are  around  7-10  g/L  and  70-80%  respectively.  

 

2.4.2 Dewatered  Sludge  Dewatered   sludge   is   the  waste  activated   sludge  with   less  water   content.  The  water   is  removed   from   the   excess   sludge   before   storage   at   anaerobic   conditions   to   avoid   the  hydrolysis   process.   Anaerobically   stored   dewatered   sludge   has   proven   to   enhance  biodegradability   due   to   earlier   breakdown   of   polyacrylamides   (PAM)   to   soluble  substrates   during   anaerobic   storage,   with   the   anaerobic   storage   acting   as   a   pre-treatment   technique   (Xu   et   al.,   2010).   Dewatered   sludge   used   in   this   work   was  dewatered  waste  activated  sludge.  

 

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2.4.3 Digested  Sludge  Digested   sludge   is   the   outcome   of   tertiary   treatment   of   sewage   treatment   plants.   The  digested   sludge   has   reduced   mass,   odour   and   pathogens   due   to   complete   anaerobic  digestion  of  primary  and  secondary  sludge  (Ek,  2005).  The  TS  and  VS  are  around  20-40  g/L  and  50%  respectively  and,  digested  sludge  had  showed   improved  dewaterability  after   thermal   and   alkaline   pre-treatments   than   conventional   process   (Carballa   et   al.,  2009).  

 

2.4.4 Fibre  Sludge  Fibre   sludge,   a   waste   material   generated   from   lignocellulosic   bio   refineries,   such   as  paper  and  pulp  industries.  These  wastes  are  either  dumped  into  the  soil  or  burnt  out,  causing   environmental   pollution   and   hence,   these   wastes   can   be   used   for   biogas  production  because  of   their  high  polysaccharide  and   low   lignin   content   (Cavka  et  al.,  2010).  

 

2.4.5 Hay  Besides  their  use  as  animal  fodder,  hay  can  be  also  for  biogas  production.  However,  its  use  for  biogas  should  be  controlled  to  protect  the  biodiversity  and  the  methane  yield  of  255-327  mL/g  VS   from   hay  of   size   range  of   0.5‒20  mm  (Stewart   et   al.,   1984).  Menind  and  Normak  (2009)  found  a  negative  correlation  between  biogas  yield,  particle  size  and  lignin  content  during  grinding  pre-treatment  of  hay.        

 

2.4.6 Maize  Silage  Maize   silage,   an   animal   fodder,   can   be   an   ideal   substrate   for   anaerobic   digestion  because   of   high   carbohydrate   and   low   lignin   content.   The   presence   of   rapidly  degrading   organic   content   leads   to   initial   increase   in   biogas   production,   which   may  limit   the   loading   rate.   Anaerobic   digestion   of   maize   silage   (30.8%   TS,   94.1%   VS)   at  mesophilic  temperature  yielded,  0.347  m3  CH4/kg  TS  ( ).  

 

2.4.7 Minced  Meat  Meat  and  other  animal  by-products  are  likely  to  be  potential  biogas  producers  because  of  the  high  fat  and  protein  content.  Recent  studies  on  anaerobic  co-digestion  of  animal  by-produc   shown  improved  methane  production   (Luostarinen  et  al.,  2009,  Luste  and  Luostarinen,  2010).  Thermochemical   pre-treatment   (   (Wu   et   al.,   2009)   of   animal   by-products  have  enhanced  the  efficiency  of  AD  process  while  pasteurization,  sterilization  

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and   alkali   hydrolysis   showed   no   improvement   in   methane   production   (Hejnfelt   and  Angelidaki,  2009).    

 

2.4.8 Orange  Peel  A   large   amount   of   solid   wastes   from   the   fruit   processing   industries   are   commonly  landfilled  because  to  avoid  the  expensive  treatment  of  these  wastes.    One  such  waste  is  the  Orange  Peel,  a   lignocellulosic  biomass   (cellulose   (%):  13.61±0.6,  hemicellulose   (%):  6.10±0.2,  lignin  (%):2.10±0.3)  (Ververis  et  al.,  2007),  containing  high  organic  content  (ca  90-95%  TS)  which  makes   it  a  suitable   feedstock   for  anaerobic  digestion.  However,   the  antimicrobial  agents  (peel  oil  and  limonin)  may  inhibit  the  digestion  process  (Naparaju  and  Rintala,  2006).      

 

2.4.9 Seaweed  Seaweed   is  multicellular  marine  algae,  which  cause  social  problems   in  coastal   regions  due   to   its  high   accumulation   resulting   from   marine   eutrophication.   Results   on   biogas  production  from  seaweeds  in  laboratory  tests  at  mesophilic  (Moen  et  al.,  1997,  Kerner  et  al.,   1991)   and   thermophilic   conditions   (Hansson,   1983)   have   been   reported.   Nkemna  and  Murto  (2010)  reported  the  effect  of  heavy  metals  removal  from  seaweed  on  biogas  production   in   batch   tests   and   UASB   reactors.   Mussgnug,   et   al.   (2010)   investigated   six  Germany  dominant  microalgae  species  (cyanobacteria,  freshwater  and  saltwater  algae)  for  biogas  production  with  drying  pre-treatment  and  they  also  concluded  that  a  suitable  cell   disruption   method   is   of   great   importance   to   enhance   the   biogas   production.   The  seaweed   used   in   this   study   was   filamentous   red   alga   of   genera   Polysiphonia,  Rhodomela  and  Ceramium.  

 

2.4.10    Yeast    Yeast   residue,   a   solid   waste   from   beer   brewery   industries   can   be   considered   as   a  suitable  substrate  for  biogas  production  because  of  its  high  organic  content.  Yeast  cells  have   a   rigid   cell   wall   constituting   mainly   of   polysaccharides,   namely   glucans   and  mannans,   which   has   to   be   ruptured   to   make   the   cell   constituents   accessible   for  anaerobic   digestion.   Cell   wall   lysis   may   be   achieved   by   pre-treatment   methods   like  enzymatic  pre-treatment  (Mallick  et  al.,  2010),  horizontal  bead  mill  (Heim  et  al.,  2007),  autolysis(Shotipruk   et   al.,   2005)   and   a   combination   of   enzymatic   pre-treatment   with  high   pressure   homogenizer   (Baldwin   and   Robinson,   1994)this  thesis  work.  

 

     

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3 Methods  and  Materials  3.1 Cellruptor  The  Cellruptor  equipment  used  in  this  work  was  obtained  from  Eco-Solids  International  Ltd.   (Hampshire,  UK)  and   is   shown   in   Figure  2.  The  assembled  equipment   consist  of  three  main  units:  8  L  pressure  cylinder  with  sampling  and  drain  ports,  compressed  CO2  tank  air   cylinder  and  a  collecting  bucket.   In  order   to  ensure   that   the   right  pressure   is  maintained   in   the   cylinder,   there   was   an   extra   gas   meter   attached   near   the   sampling  port  of  the  high  pressure  cylinder,  apart  from  the  gas  regulator  near  the  gas  cylinder.  

 

3.1.1 Process  conditions  The  pressure   that  was  operated  during   this   thesis  work  was  10  bar  and   the   residence  time   ranged   from   30   to   180   min.   The   equipment   was   handled   according   to   the  

-Solids   International   Ltd.,   Hampshire,   UK).   All   pre-treatment  run  took  place  at  room  temperature  and  the  working  volume  for  every  run  of  pre-treatment  was  between  1.5-2.0  L.                

                                                                             

 Figure  2:  Cellruptor  

           

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3.2 Batch  Experiment    3.2.1 The  substrates  &  Experimental  set-up    Ten  different  substrates  were  studied  in  five  sets  (A-E)  of  batch  experiments  and  Table  2  summarizes  all  the  substrates  with  their  collection  place  and  solids  content.  Both  batch  sets   A   and   B   utilized   biosludge   from   wastewater   treatment   plants   (WWTP),   but   the  difference   lies   in   the  aim  of  each  one.  Batch  set  A  was  designed  to  study   the  effect  of  residence   time   (30,   60,   90,   120   and   180   min)   while   batch   set   B   aimed   to   evaluate   the  effect   of   substrate   release   (rapid/non-rapid)   from   cylinder   draining   port   on   biogas  production.   Remaining   batch   sets   C,   D   and   E   were   designed   to   evaluate   the   effect   of  Cellruptor   pre-treatment   of   various   other   substrates   on   biogas   production,   under  operating  conditions  of  10  bar  pressure  and  90  min  residence  time.  

Substrates  as  starting  material  for  pre-treatment  

Cellruptor   needs   the   substrates   in   slurry   form   and   hence,   substrates   with   high   TS  content  (>10%)  and  in  non-slurry  form  were  diluted  with  water  or  digested  sludge.  The  non-treated   substrates   (or   control)   were   also   diluted,   as   to   eliminate   the   influence   of  dilution  factor  and  pre-wetting  over  biogas  production.    Few  substrates  were  processed  in  the  following  way  prior  to  Cellruptor  pre-treatment:  dry  substrates  (maize  and  hay)  were  manually  scissored  into  small  pieces  (~1-2  cm);  orange  peel  was  mashed  using  a  food  processor;  seaweed  was  initially  washed  with  water  to  remove  sand  particles  and  then  scissored  to  shorter  fragments  (~2-3  cm).    

 

3.2.2 Batch  start  up  The   mandatory   solutions   in   all   batch   bottles,   irrespective   of   the   substrate   type,   are  inoculum,  nutrient  solution,  Na2S  solution  and  Milli-Q  water.    Inoculum  was  prepared  by   mixing   the   digested   sludge   from   Nykvarn   sewage   treatment   plant   (Linkoping,  Sweden)  with   the  sludge  collected   from  various  semi-continuous  stirred   tank  reactors  (CSTR)  at  Scandinavian  Biogas  Fuels  AB.  The  nutrient   solution  comprising  of  NH4Cl,  NaCl,   CaCl2.2H2O   and   MgCl2.6H2O   while   Na2S   solution   (0.1   M)   acts   as   a   reducing  agent   ensuring   low   redox   potential   by   complete   removal   of   residual   oxygen.   Milli-Q  water   is   the   double   distilled   water   prepared   from   the   Millipore   System   (Millipore,  Billerica,  USA).  The  amount  of   loading  substrate  and  milli-Q  water   in  each  bottle  was  calculated  based  on  OLR  and  assumed  methane  potential  of  each  substrate.  

Batch  experiments  were  carried  out  in  triplicates  of  320  mL  glass  bottles  holding  100  mL  liquid   phase   and   the   procedure   were   accordant   with   Scandinavian   Biogas   Fuels   AB  standard   procedure   described   below.   The   substrates   were   weighed   and   loaded   into  their   respective   labelled  glass  bottles,  which  was   then   followed  by   flushing  N2   gas   to  secure  anaerobic  environment  in  these  bottles.  20  mL  inoculum,  2  mL  nutrient  solution  and  milli-Q  water  were  added  to  these  bottles  while  flushing  N2  gas.  The  bottles  having  been   sealed   immediately   with   EPDM   rubber   stoppers   and   aluminium   caps,   the   gas  

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phase   was   then   altered   by   evacuating   and   refilling   using   nitrogen/carbon   dioxide  mixture  (N2/CO2  ;  80/20%)  for  more  than  nine  times.  Finally,  0.3  mL  Na2S  solution  was  injected  after  depressurizing   the  bottles   completely.  The  bottles  were   then   shook  well  

 

Three   standard   controls   were   used   for   each   batch   set   and   they   were   prepared   (in  triplicates)  as  mentioned  below:  

Inoculum  Control   To   determine   the   methane   production   from   inoculum   alone   and  this   value   helps   for   the   calculation   of   methane   production   from  solely   substrate   in   substrate   bottles.   (20   mL   inoculum,   2   mL  nutrient  solution,  0.3  mL  Na2S  solution  and  78  mL  milli-Q  water)  

Positive  Control  

 

To   determine   the   degradation   efficiency   of   inoculum   by   using  cellulose   filter   paper.   (0.5   g   Whatman   filtration   paper   Grade   3  (Whatman  Ltd.,  UK),  20  mL  inoculum,  2  mL  nutrient  solution,  0.3  mL  Na2S  solution  and  78  mL  milli-Q  water)  

Methane  Control  

 

To   determine   the   instrument   reliability   by   estimating   the   known  methane  amount.  (50  mL  methane  and  100  mL  milli-Q  water)  

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Table  2:  Sample  collection  and  pre-treatment  conditions  of  various  substrates  

 

Batch  set  

 Substrate     Collection  place  in  Sweden  

Solids  content#   Pre-treatment  level  Residence  time  (min)  

Pressure  (bar)  Rapid/Non-rapid  

release##  

TS    (%)  

VS  (%TS)  

A  

Biosludge   Henriksdal  WWTP,  Stockholm.    4.3   71.0   30,60,90,120  &  180  min  10  bar  

Non-rapid  Biosludge   Municipal  WWTP,  Varberg.    5.3   78.9  

B   Biosludge   Municipal  WWTP,  Varberg.    5.2   78.6  90  min  10  bar  

Rapid  and  Non-rapid    

C  

  Supermarket,  Linkoping.   28.1   93.4  90  min  10  bar  Rapid    

Minced  meat**   Supermarket,  Linkoping.   40.5   97.5  Digested  sludge   Nykvarn  sewage  treatment  plant,  Linkoping.    2.7   66.9  Fibre  sludge*   Husum  Pulp  Plant,  Husum.   29.4   66.4  

D  

Hay*   Haga  Farm,  Östergötland.   93.3   90.9   90  min  10  bar  Rapid    

Dewatered  sludge*   Loudden  WWTP,  Stockholm.   24.7   83.8  Maize  silage*   Hags  Farm,  Östergötland.   35.2   96.8  

E  

Biosludge   Bromma  WWTP,  Stockholm.    6.2   68.2  

90  min  10  bar  Rapid    

Dewatered  sludge*   Henriksdal  WWTP,  Stockholm.   27.7   60.4  Minced  meat*   Supermarket,  Linkoping.   40.5   97.5  Biosludge   Frövi  Pulp  Plant,  Frövi.    7.1   78.3  Seaweed*   Kattegat  coast,  Varberg.   12.0   70.0  Orange  peel*   Brämhults  Juice  Industry,  Boras.   20.7   95.6  

Diluted  with  (*water/**digested  sludge)  before  pre-treatment          #  solids  content  of  fresh  substrate              ##  substrate  release  from  draining  port  of  Cellruptor  

 

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3.2.3 Analysis  Solids  content  (TS  &  VS)  

TS   and   VS   were   estimated   according   to   the   standard   protocol   of   Swedish  Standards   Institute.   A   small   amount   of   substrate   were   placed   in   a   silica  crucible  and  dried   in  hot  air  oven  at  105 C   for  20  hours.  The  crucibles  were  then  procedure  was  performed  in  duplicates  to  obtain  concordant  values.    

                                         

Where      A  -  weight  of  silica  crucible    

                             B  -  weight  of    

                             C  -  weight  of  silica  crucible  and  substrate,  after  drying    

 

in  a  muffle   furnace.  The  crucible  was  weighed   in  a  matter   chemical  balance  after  cooling  to  room  temperature.  

 Where      A  -  weight  of  silica  crucible    

                             C  -  weight  of  silica  crucible  and  substrate,  after  drying    

                             D  -  weight  of  silica  crucible  and  substrate,  after  ignition    

 

Biogas  production  

The  amount  of  biogas  produced  in  each  batch  flasks  were  evaluated  from  the  gas  pressure  measured  by  testo  digital  pressure  meter  (Testo  AG,  Lenzkirch,  Germany)  on  7  occasions  (Day  1,  3,  7,  14,  20,  31  and  60).  Having  measured  the  gas  pressure,  1  mL  of  biogas  was  withdrawn  from  the  headspace  of  each  flask  and  injected  into  their  corresponding  31.7  mL  glass  vial  for  further  analysis  by  gas   chromatography   (GC).   Then,   the   bottles   were   completely   depressurized  expect   methane   control   flasks.   For   methane   control   flasks,   pressure   is  measured  only  on  first  occasion  while  gas  sampling  is  done  on  all  occasions  together  with  other  flasks.      

Methane  content  

The   methane   content   in   the   biogas   was   determined   from   the   GC   spectra,  measured  on  a  HP  5880A  series  GC  system  (Hewlett  Packard,  Houston,  USA)  equipped  with  a  Flame  ionization  detector  (FID).  Separations  were  carried  out  by  mobile  phase  (N2  gas)  passing  through  Poraplot  T  column  at  a  flow  rate  of  130  mL/ etector  temperatures  were  80,  150  

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processing  were  executed  by  the  integrated  data  processor  (HP  5880A  series  GC  terminal).  The  methane  amount  was  interpolated  from  the  standard  curve  (methane  standards:  0.07,  0.63  and  1.71%)  and  these  standards  were  renewed  every  2  weeks.  

 

3.3 Semi-continuous  digester  experiment  3.3.1 The  substrates      Two   substrates:   biosludge   and   reject   water   from   dewatered   digester   sludge  were   collected   from   Municipal   WWTP   (Varberg,   Sweden).   Biosludge   was  diluted   with   reject   water   according   to   the   organic   loading   employed   to   the  digester.    

 

3.3.2 Digester  configuration  The   digester   experiment   was   carried   out   in   a   5   L   twin-neck   glass   bottle,   at  

rubber   stopper   was   utilized   for   feeding   and   withdrawal   purpose,   while   the  wide   neck   had   a   two-holed   rubber   stopper   that   allowed   a   PVC   tube   and   a  three-bladed   propeller   to   protrude   into   the   digester.   The   geared   motor   unit  holding   the   propeller   was   controlled   by   a   timer   which   automatically   drives  the   propeller   for   15   min   per   hour   and   the   stirring   rate   was   500   rpm  throughout   the   experiment.   The   PVC   tube   was   connected   to   the   water  displacement  bottle  consisting  of  stroke  meter.  

 

3.3.3 Digester  Operation  The  digester  was  initially  inoculated  with  4  L  of  digester  sludge  from  Varberg  WWTP  (Varberg,  Sweden)  and  thereafter,   the  diluted  substrate  was   fed   into  the  digester  once  a  day.  The  4  L  active  volume  was  maintained  by  the  semi-continuous   operational   mode   involving   the   same   withdrawal   and   feeding  amount,   calculated   from   assumed   OLR   and   HRT.   Apart   from   automatic  control,   stirring   was   also   manually   switched   for   10   min,   before   and   after  feeding,   to   ensure   homogeneity   of   digester   content.   Digester   feeding  happened  on  all  days  of  the  week  while  withdrawal  took  place  on  weekdays  and   the   exempted   withdrawals   were   compensated   by   huge   withdrawal  amount  on  Mondays  of  each  week.  The  biosludge  organic   loading  increased  from  1.5  to  2.0  g  VS/L/day  during  the  initial  digestion  period  (day  1-7)  while  initial   HRT   was   20   days   and   then,   set   to   OLR   2.5   g   VS/L/day   and   HRT15  days  from  day  8.  The  total  66  days  experimental  period  was  categorized  into  two  phases:  Control  phase  (40  days)  and  Cellruptor-treated  phase   (26  days).  Untreated  biosludge  was   fed  during  the  control  phase  while  Cellruptor  pre-treated   biosludge   was   utilized   during   the   second   phase   and   the   Cellruptor  treatments  were  made  twice  a  week.  The  pressure  in  the  digester  was  reduced  due  to  regular  discharge  of  methane  produced.  

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3.3.4 Analysis    

Solids  content  (TS  &  VS)  

Solids   content   were   analysed   twice   a   week   and   the   procedure   has   been  described  (refer  3.2.2).  

Biogas  production  

Biogas  production  was  determined  everyday  by  noting  down  the  number  of  strokes  formed  on  the  stroke  meter  place  above  the  water  displacement  bottle  and  this  bottle  is  renewed  every  14  days.    

pH  

pH   of   the   digested   sludge   was   analysed   twice   a   week   using   a   polilyte   lab  electrode   (Hamilton,   Bonaduz,   Switzerland)   integrated   with   WTW   series  Inolab   pH   730   (Wissenschaftlich-Technische   Werkstätten,   Weilheim,  Germany).  

VFA  

VFA  analysis  was  carried  out  once  a  week  using  a  HP  6890  series  GC  system  (Hewlett  Packard,  Houston,  USA)  integrated  with  an  auto-sampler  controller.  Helium   served   as   the   carrier   gas   (2   mL/min)   while   the   detector   FID  comprised  of  hydrogen  (25  mL/min)  and  air  (250  mL/min).  A  starting  oven  

for   10   min.   Injecrespectively.  

Methane  content  

1  mL  of  biogas  from  the  digester  was  syringed  out  and  expelled  into  31.7  mL  glass   vial   for   GC   analysis   (refer   4.2.3   for   GC   procedure)   to   determine   the  methane  content  (%)  and  this  analysis  was  performed  twice  a  week.    

 

3.4 Software  Statistical   analyses   were   carried   out   by   IBM®   SPSS®   Statistics   software  version  19  (International  Business  Machines  Corp.,  New  York,  USA)  and  the  executed  statistical  tests  are  mentioned  below:  

One  way  ana post-hoc   test  was  used  to  compare  various  pre-treatment  levels  in  batch  sets  A  and  B.    Among  the   post-hoc  Tukey  test  due  to  the  presence  of  control  levels.  

Paired   t-test   was   performed   to   examine   the   effectiveness   of   Cellruptor   pre-treatment   on   biogas   yield   from   various   substrates   (batch   sets   C,   D   and   E).  Paired  t-test  was  considered  because  of  one  measurement  variable   (methane  

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production)  and  two  nominal  variables  (one  was  different  substrates  and  the  - -treatment).   Independent   t-test  was   conducted   to  

compare   the   biogas   yield   of   control   and   treated   phase   of   semi-continuous  digester.  An  alpha  level  of  0.05  was  considered  during  all  statistical  analyses.    

Other   calculations   were   performed   using   inbuilt   functions   and   programs  within  a  standard  Microsoft  Excel  template,  which  is  maintained  confidential  by  Scandinavian  Biogas  Fuels  AB.  

 

 

 

 

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4 Results  4.1 Batch  experiments  Methane   yields   from   all   batch   experiments   (Mean   of   triplicates   ±   standard  deviation)  are  tabulated  under  Appendix  B.  

4.1.1 Batch  set  A  Figure   3   shows   the   influence   of   residence   time   variation   during   Cellruptor  pre-treatment   on   biogas   production   utilizing   two   biosludges   (Henriksdal  WWTP   and   Varberg   WWTP).   On   day   60,   three   different   pre-treatments   of  Varberg   biosludge   (90,   120   and   180   min)   yielded   same   amount   of   methane.  Similarly,  two  flasks  of  Henriksdal  biosludge  (60  and  180  min)  yielded  closely  same  amount  of  methane.  The  results  indicate  that  the  residence  time  studied  during  pre-treatments  did  not  affect  the  biogas  yield.  

Statistical  Data  

Day  60  methane  produced   (mL  CH4/g  VS)  were   subjected   to  an  analysis  of  variance   having   six   levels   of   pre-treatment   (control,   30,   60,   90,   120   and   180  min).   Overlapping   of   variability   amongst   the   pre-treatment   levels   (Std.  Deviation)   in   Table   3   indicate   that   there   was   no   statistically   significant  difference   between   pre-treatment   levels   for   both   biosludges,   Henriksdal  biosludge   (F(5,36)=0.03,P=0.99)   and   Varberg   biosludge   (F(5,36)=0.01,P=1.00).  Further   hypothesis   testing   and   Dunnett's   C   post-hoc   test   (Appendix   C)  confirm  these  results.  

Table  3:  ANOVA  Statistical  analyses  of  Batch  set  A  results  

 Substrate   Pre-­‐‑

treatment  levels  

N   Mean   Std.  Deviation  

Std.  Error  

95%  Confidence  Interval  for  Mean  

Minimum  

Maximum  

Upper  bound  

Lower  bound  

Biosludg

e  (H

enriksda

l  WWTP

)  

Control   7   169   80   30   94   243   22   247  30  min   7   171   80   30   97   245   24   247  60  min   7   162   81   30   87   237   25   246  90  min   7   176   84   32   98   255   25   262  120  min   7   173   84   31   94   251   20   253  180  min   7   177   88   33   96   258   23   263  

Biosludg

e  (Varberg  

WWTP

)  

Control   7   167   88   33   85   249   24   262  30  min   7   179   95   35   91   267   22   276  60  min   7   175   93   35   88   262   20   272  90  min   7   176   93   35   90   262   21   272  120  min   7   173   89   33   90   256   21   267  180  min   7   177   90   34   93   260   21   267  

 

 

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 Figure  3:  Methane  yields  obtained  from  batch  set  A,  describing  the  effect  of  

Cellruptor  pre-treatments  varying  in  residence  time  over  methane  yield.  

 

4.1.2 Batch  set  B  The  substrate  release  (rapid/non-rapid)  from  the  Cellruptor  did  not  have  any  effect   on   biogas   production   (Figure   4).   There   was   no   significant   difference  between   the   control   and   the   treated   flasks.   Moreover,   the   pre-treatment   did  

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not   also   affect   the   initial   methane   production   rate.   To   further   evaluate   the  Cellruptor  pre-treatment  on  other  substrates,  twelve  different  substrates  were  studied  in  remaining  three  batch  sets.  

 Figure  5:  Error  bar  graph  of  batch  set  B  results                                                                                                                

(Error  bars:  95%  Confidence  Interval)  

Statistical  data  

An   ANOVA   test   was   carried   out   to   find   the   statistical   significant   difference  between  the  pre-treatment  levels.  There  was  no  significant  effect  of  substrates  release   (rapid/non-rapid)   on   biogas   production,   (F(3,24)=0.01,P=0.99).   The  error   bars   with   95%   confidence   level   (Figure   5)   overlap   indicating   that   the  methane   yield   was   not   significantly   different   between   the   pre-treatment  levels.    

 

4.1.3 Batch  set  C,  D  and  E  All  substrates  treated  in  batch  sets  C,  D  and  E  are  displayed  in  Figure  6  while  methane  yields  of  all  substrates  on  Cellruptor  pre-treatment  (10  bar  pressure,  90  min  residence  time)  are  tabulated  under  Table  4.    Most  substrates  showed  an   increase   in  methane  yield,  which   reveals   the  positive  effect  of  Cellruptor  pre-treatment.   Negative   effect   was   observed   in   minced   meat   diluted   with  digested   sludge   and   also   biosludge   (Frövi   pulp   plant),   while   biosludge  (Bromma   WWTP)   showed   a   little   significant   effect.   Maize,   hay,   seaweed,  dewatered  sludge  (Henriksdal  WWTP),  minced  meat  with  water  dilution  and  fibre   sludge   showed   a   significant   effect   on   pre-treatment   starting   from   the  initial  stages  of  batch  digestion  while  orange  peel  showed  a  poor  data.  

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 Figure  4:  Methane  yields  from  batch  set  B,  illustrating  the  effect  of  rapid  (R)  

and  non-rapid  (NR)  substrate  release  from  draining  port  of  Cellruptor  on  methane  yield.    

 

Table  4:  Methane  yields  from  batch  sets  C,  D  and  E  

Batch  set  

Substrate  Methane  yield  (mL  CH4/g  VS)  

Control*   Pre-­‐‑treated*  

C  

Yeast   461±24   471±15  Minced  meat    +  digested  sludge   743±7   665±18  Digested  sludge   175±2   189±3  Fibre  sludge   235±39   254±22  

D  Hay   267±17   360±57  Dewatered  sludge  (Loudden  WWTP)   161±6   165±6  Maize   297±40   342±25  

E  

Biosludge  (Bromma  WWTP)   305±21   307±13  Dewatered  sludge  (Henriksdal  WWTP)   116±12   125±5  Minced  meat   647±29   704±21  Biosludge  (Frövi  pulp  plant)   114±2   101±5  Seaweed   155±37   175±22  Orange  Peel   291±10   344±29  

*(mean    of  triplicates  ±  standard  deviation)  

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Figure  6a:  Methane  yields  from  various  substrates,  on  Cellruptor  pre-treatment  at  90  min  residence  time  and  10  bar  pressure.                                                                          

(X  axis:  days,  Y  axis:  mL  CH4/g  VS)  

   

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 Figure  6b:  Methane  yields  from  various  substrates,  on  Cellruptor  pre-

treatment  at  90  min  residence  time  and  10  bar  pressure.                                                                          (X  axis:  days,  Y  axis:  mL  CH4/g  VS)  

 

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Statistical  data  

Three  substrates  were  excluded  from  the  following  statistical  test  due  to  some  reasons:  minced  meat  with  digested  sludge  was   the  only  co-digestion  study  made   in   this   thesis   work   and   it   would   not   be   appropriate   to   include   in   the  statistical   test,   biosludges   (Bromma   WWTP   and   Frövi   pulp   plant)   were   also  excluded  as  it  was  found  that  the  Cellruptor  pre-treatment  is  ineffective  with  highly  digesting  sludge.  

The   control   and   treated   level   of   various   other   substrates   were   paired   and   a  paired  t-test  was  carried  out  to  determine  the  Cellruptor  pre-treatment  effect  on   methane   yield   from   various   substrates.   The   mean   increase   in   methane  yield   was   significantly   greater   than   zero,   proving   that   the   Cellruptor   pre-treatment   was   effective   to   increase   the   methane   yield   (paired   t(9)   =   -3.576,  P=0.006;  Appendix  C)  

 

4.2 Semi-continuous  digester  experiment  The  biogas  production,  organic  loading,  pH  and  VS  reduction  (%)  during  the  65   days   digestion   period   are   summarized   in   Figure   7.   Approximately   25%  increase   in  biogas  yield  was  observed  between  Cellruptor  treated  phase  and  control  phase  while  a  significant  increase  of  biogas  production  was  seen  from  day   57.   The   digester   responded   well   when   the   OLR   was   increased   and  maintained  at  2.0  g  VS/L.    

Statistical  data  

An   Independent   t-test   between   the   control   and   Cellruptor   treated   phase  showed   a   significant   difference   between   the   methane   yields   of   control   and  Cellruptor  treated  phase  (t(33)  =  3.97,  P=0;  Appendix  C).  The  results  suggest  that   the   Cellruptor   pre-treatment   had   significantly   increased   the   biogas  production.  

 

 

 

 

 

 

 

 

 

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Figure  7:  Organic  loading,  pH,  biogas  production  and  VS  removal  (%)  

 of  semi-continuous  digester.  The  line  (                  )  divides  the  experimental  period  into  control  and  treated  phase  

                 Control  phase    Treated  phase  

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5 .      Discussion  5.1 Batch  experiments  

5.1.1 Biosludge  

Effect  of  residence  time  variation  of  Cellruptor  

Different   residence   time   studied   (30,   60,   90,   120   and   180   min)   displayed   no  improvements   on   methane   production   of   biosludge.   Two   biosludges  (Henriksdal   WWTP   and   Varberg   WWTP)   were   evaluated   and   found  residence   time   exceeding   90   min   produced   reasonably   equal   and   higher  amount  of  methane,  which  led  to  conclude  that  90  min  was  the  sufficient  and  effective  residence  time  for  laboratory  scale  Cellruptor  used  during  the  course  of  this  thesis  work.  During  Cellruptor  pre-treatment,  diffusion  of  CO2  into  the  cell   is   ceased   when   a   dynamic   equilibrium   is   attained.   In   this   batch  experiment,  the  dynamic  equilibrium  might  have  attained  during  90  min  pre-treatment   which   resulted   in   maximum   biodegradability   at   this   operating  condition   and   therefore,   there   is   no   increase   in   biodegradability,   with   the  increase   in   residence   time   above   90   min.   In   large   scale,   the   Cellruptor  residence   time   will   vary   as   they   depend   on   TS   content   of   the   substrates  treated.   A   similar   study   on   effect   of   residence   time   during   ozone   pre-treatment   was   reported   by   Silverstein,   et   al.   (2007)   who   observed   no  significant   changes   between   different   residence   times   of   30,   60   and   90   min.  The  optimum  residence  time  may  also  vary  as  they  depend  on  substrate  type  and  its  structural  characteristics  in  full  scale  operations.  

Effect  of  substrate  release  (rapid/non-rapid)  of  Cellruptor  

Similar   to   residence   time   variation,   no   significant   difference   was   observed  with  the  effect  of  substrate  release  (rapid/non-rapid)  compared.  Non-rapid  (5  s)   explosion   might   have   led   to   the   maximum   biodegradability   of   the  biosludge   and   further   rapid   release   (1   s)   had   shown   no   improvement   in  biodegradability.  However,  rapid  release  of  substrates  was  considered  for  the  rest  for  the  pre-treatments  since  the  explosion  factor  will  occur  more  powerful  from   the   rapid   release   than   the   non-rapid   release   of   substrates   from   the  draining   port   of   Cellruptor   and   this   rapid   release   is   also   executed   in   large  scale  operations.  Previous  studies  show  a  hefty  explosion  as  the  main  criteria  for   an   effective   degradation   steam   explosion   (municipal   wastewater   sludge)  (Dereix   et   al.,   2006),   ammonia   fibre   explosion   (switchgrass)   (Alizadeh   et   al.,  2005)   and   CO2   explosion   (corn  stover   and   switchgrass)   (Narayanaswamy   et  al.,  2011).    

Effect  of  Cellruptor  pre-treatment  under  10  bar  pressure  and  90  min  residence  time  

No   significant   difference   was   observed   on   Cellruptor   pre-treatment   with  biosludges   (Bromma   WWTP   and   Frövi   pulp   plant)   as   they   decreased   the  methane   yield   by   11   and   1%   respectively   after   Cellruptor   pre-treatment.  

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When   comparing   with   previous   data   on   Varberg   WWTP,   this   biosludge  attained   55%   VS   reduction,   which   seem   quite   high   and   these   results   reveal  that  Cellruptor  pre-treatment  is  unlikely  to  be  effective  with  highly  digesting  biosludges   in   batch   experiments.   This   similar   profile   was   also   seen   with  biosludges  (Henriksdal  WWTP,  Varberg  WWTP)  in  batch  set  1.  These  results  confirm  the  above  possible  explanation,  where  Cellruptor  is  not  effective  with  highly  digesting  biosludge.  This  high  VS  reduction  is  in  agreement  with  other  pre-treatments   such   as   ultrasonic   (Apul   and   Sanin,   2010,   Tiehm   et   al.,   1997)  and   hygienisation   pre- )   (DAVIDSSON   and   LA  COURJANSEN,   2006),   which   showed   an   increase   in   methane   yield   of  biosludge  with  55.8%  maximum  VS  reduction.  

 

5.1.2 Dewatered  Sludge  Cellruptor   pre-treatment   showed   a   positive   effect   with   increase   (11%)   in  methane  yield  utilizing  dewatered  sludge  from  Henriksdal  WWTP  while  little  effect   (methane   increase   by   2%)   was   seen   with   dewatered   sludge   from  Loudden  WWTP.  It  was  difficult  to  judge  the  Cellruptor  effect  on  dewatered  sludge   from   these   contrasting   results   and   it   is   also   possible,   that   dewatered  sludge  from  other  WWTPs  would  produce  an  entirely  different  result.  Other  reported   pre-treatment   methods   using   dewatered   sludge   include   anaerobic  storage  (Xu  et  al.,  2010)  and  addition  of  aerobic  thermophilic  bacteria  (Miah  et  al.,  2005).    

 

5.1.3 Digested  Sludge  Cellruptor  pre-treatment   improved   the  methane  yield  of  digested  sludge  by  4%  over  control  flask  and  this  increase  of  methane  yield  is  quite  significant.  In  large-scale  biogas  plants,  the  digested  sludge  is  allowed  to  degas  for  few  days  before  disposal.  The  little  amount  of  biogas  produced  from  the  slow  digestion  process   is   rarely   utilized   and   hence,   implementing   Cellruptor   pre-treatment  will  improve  the  digestion  process  resulting  in  more  methane  yield  and  also  it  may  reduce  the  pathogens  and  organic  pollutants  in  the  digested  sludge.    

 

5.1.4 Fibre  sludge  A  significant   increase   in  methane  yield   (10%)  was  observed  from  Cellruptor  pre-treated   fibre   sludge.   This   result   indicates   the   significant   effect   of  Cellruptor  on  disruption  of  lignocellulosic  components  in  fibre  sludge.  There  was   a   sudden   steep   observed   on   day   30   in   the   methane   production   curve,  which  might  have  caused  from  GC  measurement  since  the  steep  was  found  in  both  control  and  pre-treated  flasks.  Working  on  a  similar  type  of  fibre  waste  (sisal  fibre  waste)  in  mesophilic  batch  reactors,  Mshandete  et  al.  has  reported  an  increase  in  methane  yield  from  size  reduction  (2  mm)  pre-treatment  (23%  increase)   (2006)   and   mesophilic   aerobic   pre-treatment   (26%   increase)   (2005)  respectively.    

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5.1.5  Hay  A  large  significant  increase  (33%)  was  observed  in  Cellruptor  treated  hay  than  control  and  the  methane  production  from  Cellruptor  treated  flask  was  360±57  mL   CH4/g   VS.   When   compared   to   other   crop   cereals,   hay   generally   yields  low  methane  due  to  its  high  lignocellulose  content  and  less  easily  degradable  carbohydrates  (Dubrovskis  et  al.,  2009).  Hence,  this  33%  increase  of  methane  yield   is   solely   considered   to   be   a   condisruption.  Co-digestion  of  Cellruptor  treated  hay  with  cattle  dung  might  be  an   interesting   approach   to   achieve   more   methane   yield   as   Jagadabhi,   et   al.  (2008)   obtained   methane   potential   of   376   mL   CH4/g   VS   when   co-digesting  grass  silage  with  cow  manure  in  batch  assays.    

 

5.1.6  Maize  Silage  Even   though   Cellruptor   treated   maize   silage   produced   a   normal   amount   of  methane   yield   (342±25   mL   CH4/g   VS),   it   was   not   possible   to   evaluate   the  Cellruptor   effect   due   to   experimental   failure   incurred   by   one   of   the   control  flask   yielding   low   amount   of   biogas.   Recalculation   (data   not   shown)   after  ignoring   the   methane   values   from   the   failed   control   flask   registered   a   large  increase   (157%)  of  methane  yield   than  control.  The  reported   increase   (157%)  due   to   effective   disruption   of   fibres   of   maize   silage   by   Cellruptor,   is   quite  higher   than   the   result   from   ultrasonication   pre-treatment   which   increased  methane  yield  of  maize  silage  by  16.9-29.5%  (Závacký  et  al.,  2010).  Due  to  lack  of   time,   the   experiment   was   not   repeated   but   a   similar   substrate   category,  maize  stalks  were  pre-treated  with  Cellruptor,  as  a  part  of  R&D  activities  of  Scandinavian  Biogas  Fuels  AB,  which  revealed  a  massive   increase   (116%)   in  methane  yield  over  control  for  35  days  anaerobic  digestion  (data  not  shown)  while   the   methane   yield   after   35   days   of   batch   digestion   from   the   treated  maize  stalks  was  335±23  mL  CH4/g  VS.    

 

5.1.7 Minced  Meat  Cellruptor   treated   minced   meat   resulted   in   higher   methane   yield   (8%)   than  the  untreated  meat,  which  may  be  due,  to  the  fact  that  the  pre-treatment  have  enhanced   the   hydrolysis   of   fatty   meat   tissues.   The   methane   yield   from  Cellruptor   pre-treated   meat   was   704±3.00   mL   CH4/g   VS   and   this   value   is  higher  when  comparable  to  methane  yield  from  thermophilic  batch  digestion  of   pork   meat   (550-550   mL   CH4/g   VS)   (Hejnfelt   and   Angelidaki,   2009).   The  above   comparison   shows   that   the   mesophilic   temperatures   may   be   suitable  than   thermophilic   temperatures   concerning   digestion   of   animal   by-products  (ABP),  which  was  earlier  reported  by  Angelidaki  and  Ahring  (1993).  With  the  thesis  data  on  co-digestion  of  minced  meat  with  digested  sludge,   theoretical  increase   in   methane   yield   solely   from   Cellruptor   treated   minced   meat   was  calculated.   The   theoretical   increase   in   methane   yield   (7%)   is   approximately  representing   the   above   mentioned   8%   methane   increase,   verifying   the  

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Cellruptor   pre-treatment.   This   was   not   the   same   case   with   other   pre-treatments  as  a  significant   increase  of  methane  yield  was  not  observed  from  

pre-treatment  (Hejnfelt  and  Angelidaki,  2009).    

 

5.1.8 Co-digestion  of  minced  meat  with  digested  sludge    The  results  obtained  from  this  co-digestion  could  not  be  discussed  because  the  control   flask   prepared   was   misappropriated.   However,   Co-digestion   of  digested   sludge   with   minced   meat,   together   with   Cellruptor   pre-treatment  was  observed  feasible  due  to  high  amount  of  methane  produced  (665±18  mL  CH4/g  VS).  In  this  co-digestion  process,  control  flask  produced  more  methane  yield  than   the  Cellruptor   treated   flask.  This  might  be  due  to   the  presence  of  high  amount  of  digested  sludge  in  control  flask  used  for  dilution,  which  acted  as   an   additional   inoculating   medium   during   the   batch   process.   It   is   also  important   to   determine   the   optimum   feed   ratios   (Luste   and   Luostarinen,  2010)   before   co-digesting   two   substrates   for   achieving   high   methane  production,  which  was  not  assessed  in  this  thesis  work  due  to  time  constraint.    

 

5.1.9 Orange  Peel  There  was  a  major  complication   in   interpreting  data   from  orange  peel  batch  results   due   to   the   observation   of   a   declined   lag   phase   followed   by   an  exponential   phase.   The   declined   lag   phase   during   initial   days   of   digestion  may   be   probably   due   to   substrate   overloading   (data   not   shown)   in   batch  flasks.   Other   possible   explanation   might   be   the   presence   of   inhibitory   anti-microbial   substances   which   normally   affect   the   anaerobic   digestion   of   fruit  and  vegetable  wastes  (Martín  et  al.,  2010).    The  exponential  phase  started  from  day  14   for  both   treated  and  control   flasks,  and   thereafter,  Cellruptor   treated  flask  yielded  more   methane   than   the   control   flask.  However,   concluding  on  the   Cellruptor   effect   of   orange   peel   is   difficult   due   to   inconsistent   results.  Further  studies  will   likely  be  needed  to  evaluate  Cellruptor  effect  on  orange  peel.    

 

5.1.10  Seaweed  Seaweed   showed   12%   significant   increase   in   methane   yield   after   Cellruptor  pre-treatment.   The   observed   steady-state   rate   of   methane   production   shows  that   the  digestion  process  was  not   inhibited  by   the  presence   of  polyphenols  and  divalent  ions,  which  may  affect  the  digestion  process  (Moen  et  al.,  1997).  The   seaweed   used   in   this   thesis   work   was   filamentous   red   alga,   and   the  methane  production  is  dependent  on  algal  species,  while  the  highest  methane  producer   was   green   freshwater   alga   Chlamydomonas   reinhardtii   (387.4±5.8  mL  CH4/g  VS;  (Mussgnug  et  al.,  2010).  Other  pre-treatments  such  as  thermal  pre- (De   Schamphelaire   and   Verstraete,   2009)   and  drying  (Mussgnug  et  al.,  2010)  showed  no  increase  of  methane  yield.    

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5.1.11    Yeast  Cellruptor  pre-treated  yeast  cells  yielded  significant  5%   increase   in  methane  production   than   control   due   to   effective   disruption   of   intact   yeast   cell   wall.  This   result   seems   to   be   interesting   when   comparing   with   other   pre-treatments,   where   Neira   and   Jeison   (2010)   found   no   significant   increase   in  biogas   yield   while   investigating   the   anaerobic   co-digestion   of   yeast   residue  with   brewery   wastewater   in   batch   and   UASB   reactor   experiments  implementing  three  pre-treatments  (thermal,  chemical  and  mechanical).  

 

 

5.2   Semi-continuous  digester  experiment  5.2.1 Biogas  production  and  Methane  content  Cellruptor  pre-treatment  produced  a  significant  increase  in  biogas  production  during   the   66   days   digester   experiment.   The   biosludge   obtained   at   6  consecutive   occasions   from   Varberg   WWTP   showed   many   fluctuations  during  the  study  period  where  the  VS  contents  ranged  from  40.81  to  62.78  g  VS/kg.  The  average  specific  biogas  yield  of  untreated  and  Cellruptor  treated  biosludge   was   223   and   273   mL/g   VS   respectively,   which   shows   22.4%  increase   in   biogas   yield.   Comparing   with   other   pre-treatments,   (Apul   and  Sanin,   2010)   indicated   55%   increase   in   biogas   yield   from   ultrasonicated  biosludge  during  mesophilic   semi-continuous  digestion  process   (SRT:  7.5  D,  OLR:  0.5  kg/L  d).  The  methane  content  in  the  Cellruptor  treated  phase  ranged  between  53-66%  over  organic  loading  rate  of  2g  VS/L  d.      

During  Cellruptor  phase  (day  41-66),   there  were  some  fluctuations  observed  with   biogas   production   rate   while   the   main   fluctuation   was   the   increase   in  biogas  production  from  day  58.  The  average  biogas  yield  between  Cellruptor  period  I  (day  41-57)  and  period  II  (day  58-66)  was  different,  and  the  respective  values  were  236  and  342  mL/g  VS.  This  increase  in  biogas  was  in  agreement  with   increase   in   VS   reduction   from   31.66(±1.24)   to   35(±1.63)%   (period   I   to  period   II).   The   enhanced   biogas   production   could   also   be   explained   by   the  fact  that  diluted  feeding  that  started  from  day  54  has  improved  the  activity  of  hydrolytic  bacteria  and  this  seems  logical  with  the  decrease   in  TS  content  of  the   digester   and   increase   in   VS   reduction   percentage.   Complicating   the  evaluation  is  a  concomitant  change   in  gas  meter   for   the  reactor  which  could  have  led  to  a  higher  measured  gas  production  but  there  were  no  indications  for   that   particular   gas   meter   to   have   malfunctioned.   Semi-continuous  digestion   of   Cellruptor   treated   biosludge   during   period   II   after   17   days  (period  I)  was  too  short  to  bring  out  strong  conclusions.  

Batch   experiments   are   generally   considered   to   screen   the   substrates   for  continuous  process  as  the  outcome  of  continuous  process  could  be  predicted  from  batch  results.  Working  on  biosludge  from  Varberg  WWTP,  batch  results  did  not  show  significant  differences  on  Cellruptor  pre-treatment  while  semi-continuous   digester   showed   a   possible   improvement.   One   reason   might   be  

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the   inoculum   has   not   adapted   to   the   pre-treated   biosludge   in   the   batch  experiments.  Another  could  be  the  dilution  of  the  sludge  in  the  batch  methods  enhancing  accessibility  and  gas  production.  As  discussed  earlier,  the  high  VS-reduction  (ca  55%)  in  the  batch  experiment  might  have  decreased  the  effect  of  the  Cellruptor  treatment  while   the   lower  VS-reduction  in  the  CSTR  (ca  33%)  leaves  room  for  enhancement.  

 

5.2.2 pH,  VFA  and  VS  reduction  The  pH  value  in  the  digester  was  between  7.4-7.6  for  the  whole  experimental  period,   reflects   the   constancy   of   anaerobic   digestion,   which   is   in   clear  agreement   with   the   review   of   (Mata-Alvarez   et   al.,   2000)   showing   good  production   rate   of   methane   for   biosludges   with   high   moisture   content   (90-96%)  in  pH  range  6.6-7.8.  The  process  stability  was  distinctly  indicated  by  the  presence   of   low  concentration  of   acetic   acid   (below  0.6  mM)  and  absence  of  propionic  acid.  Earlier  studies  report  that  the  concentration  of  individual  VFA  below   50   mM,   do   not   inhibit   the   anaerobic   digestion   process   (Ahring   et   al.,  1995,  Pullammanappallil  et  al.,  2001).  The  average  VS  reduction  over  control  and   Cellruptor   phase   were   33.54(±2.01)   %   and   33.33(±2.21)   %   respectively.  These  values  fall  within  range  32.7  to  34.4%,  which  was  found  in  the  literature  describing   acid   pre-treatment   of   biosludge   (Devlin   et   al.,   2010).   This   slight  increase  of  VS  reduction  may  be  due  to  the  poor  degradability  of  biosludges  with  high  sludge  age  (15-20  days)  (Dwyer  et  al.,  2008).  

 

5.3 Evaluation  of  Cellruptor  Pre-treatment  The  methane  yields  from  control  and  treated  flasks  are  not  enough  to  evaluate  a   pre-treatment   technique   since   these   methane   yields   constitute   basic  information   to   identify   suitable   substrates,   while   further   analysis   of   cost  expenses  and  payback  time  should  be  calculated,  and  these  improvements  are  likely   needed   to   harness   the   potential   of   Cellruptor   in   enhancing   biogas  production.  

On  one  side,  being  a  new  technology  with  no  relevant  literatures  available,  it  was  difficult  to  evaluate  the  Cellruptor  effect.  On  the  other  side,  pre-treatment  tein  methane  yield.  Since  economic  and  environmental  prospects  are  also  to  be  considered,  energy  consumption  by  these  pre-treatment  techniques  must  also  be  compared  to  find  an  effective  pre-treatment  technique  for  every  substrate.    This  thesis  work  has  many  shortcomings,  but  it  does  seem  to  demonstrate  that  the  Cellruptor  pre-treatment  was  found  to  be  effective  in  increasing  the  biogas  production   especially   with   dry   substrates,   such   as   maize,   hay,   etc.,   than  

-treatment  is  effective  to  enhance  the  hydrolysis  of  lignocellulosic  biomass  since  it  might  be  because  of  weak  carbonic  acid  formed  in  the  Cellruptor  from  CO2  gas  and  moisture   content   of   the   substrate.   Before   starting   batch   experiment,   treated  

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substrates  after  Cellruptor  pre-treatment  were  stored  in  the  refrigerator  (less  than  18  hours)  and  at  low  temperatures,  weak  carbonic  acid  formed  enhances  the   hydrolysis   of   cellulosic   biomass   (Narayanaswamy   et   al.,   2011,   Peter   van  Walsum  and  Shi,  2004).      

The  results  reported  in  this  thesis  work  are  preliminary  and  these  results  may  vary   within   same   feedstock   as   their   characteristics   change   based   on   their  geographical  location.  Undeniably,  further  investigations  on  enhancing  biogas  production   from   various   waste   substrates   by   Cellruptor   pre-treatment   may  also  provide  a  solution  to  serious  environmental  threats  like  ocean  and  river  dumping,   and   land   filling.   The   scalability   of   Cellruptor   could   also   be   of  significant   interest   to   biogas   companies   focused   on   increasing   biogas  production.  

 

 

 

 

                                                                                                                                       

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6 .   Conclusion    

Cellruptor  pre-treatment  improved  anaerobic  digestion  process  leading  to  an  increase   in   biogas   production.   Positive   results   were   reported   on   most   feed-stocks   tested  using   the  BMP  (batch)   tests  with  exception  of  biosludge.   Batch  experiments  showed  a  notable  increase  in  methane  yield  in  seaweed,  hay  and  fibre   sludge   on   Cellruptor   pre-treatment,   while   the   pre-treatment   had   little  effect   on   methane   yields   of   biosludge   and   dewatered   sludge.   However,   the  results   from   semi-continuous   digester   indicate   that   the   Cellruptor   pre-treatment   has   improved   the   biogas   production   of   biosludge   by   22.4%.  Unfortunately,   it   was   not   possible   to   evaluate   the   pre-treatment   effect   on  maize  and  orange  peel  due  to  experimental  failure.    

Eco-Solids   International   Ltd.   (Hampshire,   UK)   has   demonstrated   a   reliable  and   commercially   viable   increase   of   methane-rich   biogas   generation   from  bench-scale,   full-scale   pilot   plant   and   commercially   operational   units   of  Cellruptor,   using   biosludge   from   municipal   sewage   treatment   plants.     It   is  recognised   that   elements   of   the   work   undertaken   may   not   fully   reflect   the  results  attainable  at  full-scale,  over  prolonged  periods  of  operation.  This  thesis  was   conducted   over   a   short   time   and   is   a   preliminary   work   in   progress.    Further   secondary   studies   are   required  and  elements  of   the  work   should  be  repeated,   in  order  to   implement  this  simple  and  low  energy  consuming  pre-treatment   technique,  as  a   reliable  and  cost-effective  substitute   for  enhancing  biogas  yield  in  large-scale  companies.  

 

 

 

 

 

 

 

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ACKNOWLEDGEMENT    

This   thesis   work   has   provided   me   an   exciting   and   rewarding   experience  which   would   not   have   been   possible   without   the   support   and   guidance   of  many  intelligent  and  fantastic  people.  

First  and  foremost,  I  am  truly  grateful  to  my  supervisor,  Andreas  Berg,  for  all  his   support   in   a   number   of   ways   throughout   this   work.   I   would   not   have  imagined  a  better  supervisor  and  mentor  for  my  thesis.  

I   owe   my   sincere   gratitude   to   the   following   people   at   Scandinavian   Biogas  Fuels  AB  

              Jörgen  Ejlertsson  for  offering  me  an  opportunity  to  do  my  thesis  at  the  company      

              Xu-Bin   Troung,   Lina   Cardell,   Björn   Magnusson,   Carina   Almé,   Ida  Andersson   and   Mia   Wirf*   for   advising   and   sharing   their   professional  experience  with  me  

I  would  also  like  to  thank  

               Newman  from  Eco-Solids  International  Ltd.,  for  guiding  with  the  installation  of  the  Cellruptor  equipment  

               

And,   finally   a   BIG   thanks   to   my   mom   and   dad   for   their   never-ending   love,  support  and  encouragement  for  all  these  years.  

   

 

 

 

 

 

 

 

 

 

 

 

*presently  at  Swedish  Geotechnical  Institute  

43  

 

APPENDIX  A:  Statistics  Sweden    

 

Figure  8:  Deliveries  of  the  vehicle  gas  (biogas  and  natural  gas)  in  Sweden,  Volume  Nm3  

44  

 

APPENDIX  B:  Batch  raw  data  

 Table  5A:  Methane  Yield  (mL  CH4/g  VS)    from  batch  experiments  

    Substrate   Day           1   3   7   14   20   31   60  

Batch  set  A  

Positive  Control   1   58   250   394   392   396   393    (Whatman  paper)                                               1 5 25 37 30 30 32

Biosludge  (Henriksdal  W

WTP)  

Control   22   106   161   198   216   235   247       1 4 2 2 1 4 3

 30  min   24   107   169   197   221   234   247       1 4 5 6 5 6 5

 60  min   25   87   145   192   213   227   246       2 24 10 15 18 28 19

 90  min   25   111   160   205   230   244   262       0 2 3 4 3 3 5

 120  min   20   106   161   203   229   239   253       1 5 3 7 3 1 4

 180  min   23   106   160   203   230   258   263       1 7 7 13 11 15 13

Biosludge  (Varberg  WWTP)  

 Control   24   81   146   193   216   251   262       3 10 10 19 19 22 24

 30  min   22   93   155   207   229   274   276       2 2 3 11 8 12 13

 60  min   20   91   150   204   221   272   269       1 6 5 11 9 13 11

 90  min   21   95   151   205   226   272   267       0 1 2 7 6 10 11

 120  min   21   97   148   204   223   255   267       1 0 3 11 5 7 8

 180  min   21   95   165   210   225   256   267       0 1 8 10 13 13 17

Batch  set  B  

Positive  Control   7   86   282   352   358   362   367    (Whatman  paper)                                                   0 2 4 5 4 7 7

Biosludge          (Varberg  

WWTP)  

 Control   21   106   157   204   217   231   255       2 2 2 4 6 4 5

 30  Min  Rapid   20   107   156   204   216   227   250       2 6 3 7 4 3 4

 180  Min  Rapid   23   109   165   211   225   241   262       1 1 4 3 4 1 1

30  Min  Non-­‐rapid   24   109   164   205   219   234   259       2 5 4 4 4 6 5

  standard  deviation(abs)  

45  

 

 

 

Table  5B:  Methane  Yield  (mL  CH4/g  VS)  from  batch  experiments       Substrate   Day           1   3   7   14   20   31   60  

Batch  set  C  

Positive  Control   1   68   195   357   361   371   402  (Whatman  paper)                                                       1 1 24 10 11 10 11 Yeast  :  Control   31   228   387   433   439   438   461  

    1 12 27 28 21 21 24 Yeast  :  90    Min   33   257   400   451   471   459   471  

    2 9 10 17 5 16 15 Fibre  sludge  :  Control   6   64   147   208   224   216   235  

    0 15 42 44 41 40 39 Fibre  sludge  :  90    Min   10   85   166   232   247   236   254  

    1 1 9 15 17 20 22 Minced  meat  :  Control   28   177   509   729   713   722   743  

    2 5 22 5 6 10 7 Minced  meat  :  90    Min   79   314   558   662   634   635   665  

    2 7 19 15 15 17 18 Digested  sludge  :  Control   13   34   63   110   122   142   175  

    1 1 1 1 1 1 2 Digested  sludge  :  90  Min   14   42   72   115   126   143   189  

      2 8 2 5 2 2 3

Batch  set  D  

Positive  Control   3   77   244   340   359   372   390  (Whatman  paper)                                                       1 4 4 6 3 10 6 Hay  :  Control   17   87   144   192   219   246   267  

    1 5 11 17 19 21 17 Hay  :  90    Min   21   114   185   254   288   325   360  

    0 6 17 32 38 48 57 Dewatered  sludge  :  Control   4   20   38   62   77   108   161  (Loudden  WWTP)                                                         0 0 1 3 2 2 1 Dewatered  sludge  :  90    Min   3   21   38   63   80   109   165  (Loudden  WWTP)                                                         0 1 1 2 3 4 6 Maize  :  Control   20   19   18   58   88   189   297  

    1 2 19 60 39 87 40 Maize  :  90    Min   24   72   168   248   273   308   342  

    1 12 20 6 7 23 25

 

 

 

 

 

 

  standard  deviation(abs)  

46  

 

 

 

 

Table  5C:  Methane  Yield  (mL  CH4/g  VS)  from  batch  experiments       Substrate               Day                       1   3   7   14   20   31   60  

Batch  set  E  

Positive  Control   1   38   139   309   324   340   348    (Whatman  paper)                                                     1 4 17 15 10 16 22 Biosludge  :  Control   4   28   51   72   83   98   114  (Frövi  pulp  plant)                                                         4 2 1 2 2 2 2 Biosludge  :  90    Min   5   29   47   65   73   86   101    (Frövi  pulp  plant)                                                       1 1 2 2 2 2 5 Dewatered  sludge  :  Control   18   38   56   50   63   83   116  (Henriksdal  WWTP)                                                 0 3 6 8 8 10 12 Dewatered  sludge  :  90    Min   20   43   61   57   72   91   125    (Henriksdal  WWTP)                                               1 0 2 3 2 3 5 Minced  meat  :  Control   31   179   441   623   642   651   647  

    1 9 27 44 33 32 29 Minced  meat  :  90    Min   35   180   442   661   697   709   704  

    2 12 47 49 33 30 21 Biosludge   24   112   209   244   265   287   305  (Bromma  WWTP)                                                         0 4 13 8 7 10 12 Biosludge  :  90    Min   24   115   198   237   260   283   307  (Bromma  WWTP)                                                         1 2 11 11 9 10 13 Seaweed  :  Control   18   53   94   117   125   142   155  

    2 11 23 28 30 36 37 Seaweed  :  90    Min   21   60   103   129   139   163   175  

    1 4 9 15 14 17 22 Orange  Peel  :  Control   7   -­‐2   -­‐18   -­‐45   -­‐36   67   291  

    0 1 3 2 12 3 10 Orange  Peel  :  90    Min   9   1   -­‐15   -­‐44   17   106   344  

    2 6 2 2 25 36 29

  standard  deviation(abs)  

47  

 

 

APPENDIX  C:  Statistical  data    

Table  6:  Hypothesis  testing  for  results  from  batch  sets  A  and  B  

Batch set A Batch set B Biosludge (Henriksdal WWTP) Biosludge (Varberg WWTP) Biosludge (Varberg WWTP)

Ho: U1 = U2 = U3

Ha: At least one mean is significantly different Ha: At least one mean is significantly different Ha: At least one mean is significantly different

Group Mean Variance Group Mean Variance Group Mean Variance

Control 169.2857143 6521.904762 Control 167.5714286 7906.952381 Control 170.1428571 6796.142857

30 min 171.2857143 6438.238095 30 min 179.4285714 9056.285714 30 min R* 168.5714286 6595.285714

60 min 162.1428571 6577.47619 60 min 175.2857143 8817.904762 180 min R* 176.5714286 7210.619048

90 min 176.7142857 7175.904762 90 min 176.7142857 8674.238095 30 min NR* 173.4285714 6772.285714

120 min 173 7139 120 min 173.5714286 8087.285714 *R-Rapid , NR-Non-rapid

180 min 177.5714286 7747.619048 180 min 177 8156.333333

Alpha level= 0.05 Alpha level= 0.05 Alpha level= 0.05

Sum of Squares Within = 249600.8571 Sum of Squares Within = 304194 Sum of Squares Within = 164246

Sum of Squares Between = 1110.47619 Sum of Squares Between = 586.7857143 Sum of Squares Between = 266.1071429

Total mean = 171.6666667 Total mean = 174.9285714 Total mean = 172.1785714

Within variance MS(Error) = 6933.357143 Within variance MS(Error) = 8449.833333 Within variance MS(Error) = 6843.583333

Between variance MS(Treatment) = 222.0952381

Between variance MS(Treatment) = 117.3571429 Between variance MS(Treatment) = 88.70238095

F Test Statistic = 0.032032857 F Test Statistic = 0.013888693 F Test Statistic = 0.012961394

Critical F-Value = 2.477168673 Critical F-Value = 2.477168673 Critical F-Value = 3.00878657

P-Value = 0.999434091 P-Value = 0.999927334 P-Value = 0.997925108

Decision Do not Reject Ho Decision Do not Reject Ho Decision Do not Reject Ho

48  

 

 

Table  7 -hoc  test  for  results  from  Batch  set  A  and  B  

 

Substrate   (I) Treatment (J) Treatment

Mean Difference (I-

J) Std. Error Sig.

95% Confidence

Interval

   

 

Lower Bound

Batch  set    

A  

Bio

slud

ge

(Hen

riksd

al W

WTP

) 30 min Control 2 44.50797 0.82 -101.1586

60 min Control -7.14286 44.50797 0.877 -110.3014

90 min Control 7.42857 44.50797 0.779 -95.73

120 min Control 3.71429 44.50797 0.807 -99.4443

180 min Control 8.28571 44.50797 0.772 -94.8729

Bio

slud

ge

(Var

berg

W

WTP

)

30 min Control 11.85714 49.1349 0.752 -102.0255

60 min Control 7.71429 49.1349 0.783 -106.1684

90 min Control 9.14286 49.1349 0.772 -104.7398

120 min Control 6 49.1349 0.795 -107.8827

180 min Control 9.42857 49.1349 0.77 -104.4541

Batch  set  

B  

Bio

slud

ge

(Var

berg

W

WTP

) 30 min Rapid Control -1.57143 44.21888 0.763 -97.5088

180 min Rapid Control 6.42857 44.21888 0.695 -89.5088

30 min Non-rapid Control 3.28571 44.21888 0.722 -92.6516

a. Dunnett t-tests treat one group as a control, and compare all other groups against it.

 

Table  8:  Paired  t-test  for  results  from  batch  sets  C,  D  and  E  

Paired Samples Statistics Paired Samples Correlations

    Mean N Std. Deviation

Std. Error Mean     N Correlation Sig.

Pair 1 Control 280.5 10 162.535 51.398

Pair 1 Control

& Treated

10 0.989 0 Treated 312.9 10 175.438 55.478

Paired Samples Test

   

Paired Differences

t df Sig. (2-tailed) Mean Std.

Deviation Std. Error

Mean

95% Confidence Interval of the

Difference

Lower Upper

Pair 1 Control - Treated -32.4 28.652 9.061 -52.896 -11.904 -3.576 9 0.006

 

 

 

 

49  

 

 

Table  9:  Independent  t-test  of  results  from  digester  experimental  results  

Group Statistics

      Treatmen

t N Mean Std. Deviation Std. Error Mean

    Methane Cellruptor

treated 26 272.65 58.539 11.48

    Control 40 223.43 29.624 4.684

Independent samples test

   

Levene's Test for

Equality of Variances

t-test for Equality of Means

F Sig. t df

Sig. (2-

tailed)

Mean Differen

ce

Std. Error

Difference

95% Confidence

Interval of the Difference

Lower

Upper

Methane

Equal variances assumed

18.345 0 4.51

5 64 0 49.229 10.904 27.447

71.011

Equal variances not assumed

        3.97 33.422 0 49.229 12.399 24.01

5 74.44

3