Acidosis  induction  affects  fermentation  patterns  and  bacterial

In   the   present   work,   the   acidosis   challenge   was   induced   through   the   reduction   of   buffering  substances  in  the  artificial  saliva.  The  Illumina  sequencing  approach  revealed   a  decrease  in  the  alpha  diversity  when  the  pH  decreased  below  SARA  thresholds.  We   observed  a  decreasing  Shannon  index  and  a  lower  number  of  observed  ASVs.  This   conforms  to  in  vitro  studies  of  Colombatto  et  al.  (2003)  and  Calsamiglia  et  al.  (2002),   who   induced   SARA   in   a   dual-­flow   continuous   culture   system   by   modifying   the   concentration  of  the  buffer  solution.  Khafipour  et  al.  (2009c)  observed  the  same  effect   in  an  in  vivo  study  where  the  bacterial  diversity  decreased  during  acidosis  challenges.  

Contrarily   to   our   observations,   the   bacterial   richness   was   not   affected   by   SARA   induction;;   however,   bacterial   counts   in   treatment   groups   were   numerically   lower   compared  to  non-­SARA  groups.  

In   the   present   work,   the   abundances   of   the   main   ruminal   phyla  Firmicutes   and   Bacteroidetes  (Jami  and  Mizrahi,  2012)  were  not  affected  by  the  acidosis  induction.  

However,  we  observed  alterations  on  family  level,  where  unclassified  Bacteroidales   decreased   during   low   pH   values.   Members   of   the   Gram-­negative   phylum   Bacteroidetes   are   reported   to   be   very   pH   sensitive   and   to   decrease   during   low   pH   values  (Khafipour  et  al.,  2009c,  Plaizier  et  al.,  2012).  During  SARA  challenges,  free   bacterial   lipopolysaccharide   endotoxins   (LPS)   are   released   from   the   bacterial   membrane  due  to  pH  dependent  cellular  disintegration  and  lysis  (Wells  and  Russell,   1996).  In   vivo,   the   free   endotoxins   are   translocated   from   the   rumen   to   the   interior   circulation  (Khafipour  et  al.,  2009b,  Plaizier  et  al.,  2012).  Several  sequelae,  such  as  

laminitis  and  liver  abscesses  have  been  associated  with  an  increased  LPS  shedding   during  SARA  conditions  (Nocek,  1997,  Emmanuel  et  al.,  2008).  However,  in  this  study   we  did  not  measure  the  LPS  concentration  in  the  fermentation  vessels  during  acidosis   period.  

During   the   time   of   low   pH   values,   we   observed   a   slightly   enhanced   lactate   concentration  in  all  treatment  groups.  However,  the  concentrations  remained  low  in  all   reaction   vessels.   During   AP,   the   NGS   approach   revealed   an   increase   of   lactate   producing  Lactobacillaceae   in   groups   with   low   pH   values.   Furthermore,   families   Lactobacillaceae  and  Streptococcaceae  increased  in  control  groups  with  an  enhanced   concentrate  supply  during  acidosis  period.  Lactate  is  considered  to  be  an  intermediate   product   from   starch   and   soluble   sugar   fermentation   by   acid   resistant   bacteria,   like   Lactobacillus   spp.   and  Strepococcus  bovis  (Nagaraja   and   Lechtenberg,   2007).   The   relatively   low   lactate   levels   of   this   present   study   appear   contrary   to   the   report   of   a   previously   performed   Rusitec   experiment   by   Eger   et   al.   (2017).   Authors   reported   increasing  lactate  concentrations  up  to  11.3  mM,  while  the  pH  decreased  to  a  nadir  

<  pH  5.0.  The  low  lactate  levels  in  our  study  possibly  result  from  higher  experimental   pH  values  and  also  from  an  enhanced  lactate  utilization  by  lactate  fermenting  bacteria.  

These   bacteria   consume   lactate   to   produce   SCFA   in   pH   ranges   above   pH   5.5   (Nagaraja  and  Lechtenberg,  2007),  which  complies  with  the  observed  pH  values  during   our   acidosis   period.  Megasphera   elsdenii  is   especially   known   for   increasing   lactate   utilization  when  pH  values  decrease  (Khafipour  et  al.,  2009c,  Fernando  et  al.,  2010).  

During  low  pH  values,  Megasphera  elsdenii  converts  lactate  to  propionic  acid  (Nocek,   1997).  Khafipour  et  al.  (2009c)  observed  the  microbiome  during  SARA  periods  and   reported   that   the   growth   rate   of   Megasphera   elsdenii   was   synchronized   to   the  

increasing   abundance   of   the   lactate   producing  Streptococcus   bovis.   The   authors   concluded  that  Megasphera  effectively  eliminated  rumen  lactate.  Megasphera  elsdenii   belongs  to  the  family  Veillonellaceae  (phylum  Firmicutes)  and  in  our  study,  the  relative   abundance  of  the  family  Veillonellaceae  increased  during  acidosis  period  in  the  liquid   phase.   Another   bacterium,   which   is   related   to   pH   maintenance   is  Selenomonas   ruminantium,   (Fernando   et   al.,   2010),   which   belongs   to   the   family   of   Acidaminococcaceae.   A   subspecies   of  Selenomonas   is   known   for   lactate   utilization   during  low  pH  values.  In  the  liquid  phase,  we  observed  an  increase  of  one  ASV  during   AP  in  treatment  groups  with  a  low  concentrate  feeding.  However,  in  our  trial  the  relative   abundance   of  Acidaminococcaceae   was   unaltered   throughout   the   experiment.  As   expected,   the   degradation   of   hay   was   diminished   during   AP   in   the   present   work.  

Cellulolytic   bacteria   are   very   sensitive   to   low   pH   values   and   decrease   in   acidotic   environments  (Russell  and  Dombrowski,  1980a,  Fuentes  et  al.,  2009).  During  AP,  both   sequencing   approaches   revealed   a   diminished   abundance   of  Fibrobacteres   and   Spirochaetes   within   the   fluid   fraction   during   acidosis   challenge.   The   impaired   degradation  of  hay  reflects  the  decreasing  abundance  and  binding  capacity  of  fibrolytic   bacteria   (Russell   and   Wilson,   1996).   Furthermore,   the   phyla  Fibrobacteres   and   the   family  Ruminococcaceae  are  reported  to  be  very  pH  sensitive  (Roger  et  al.,  1990)  and   diminished   during   our   acidosis   trial   in   all   acidosis   groups.   In   our   study,   the   acetate   production   decreased   during   low   pH   values,   presumably   resulting   from   a   reduced   activity  of  fibrolytic  and  cellulolytic  bacteria  (Leedle  et  al.,  1982,  Thoetkiattikul  et  al.,   2013).   Moreover,   the   bacterium  Streptococcus  bovis  converts   glucose   to   acetate.  

However,  during  low  pH  values  or  excessive  glucose  presence  Streptococcus  bovis   switches  from  acetate  to  lactate  production,  as  the  enzyme  which  is  converting  glucose  

to  acetate  is  pH  sensitive  (Abbe  et  al.,  1982).  This  may  have  supported  the  decreasing   acetate  production  in  our  study.    

The  impact  of  low  pH  values  on  the  microbial  diversity  is  also  visible  in  the  production   pattern  of  the  SCFA.  In  a  recent  in  vivo  study,  Mao  et  al.  (2013)  reported  an  increasing   propionate  and  butyrate  production  during  SARA  conditions  (pH  <  5.8  for  5  h)  in  dairy   cows.  In  contrast  to  the  study  of  Mao  et  al.  (2013),  the  molar  proportions  of  propionate   were  not  affected  in  our  trial  and  remained  unaltered  in  most  groups.  Only  at  the  end   of  AP,  the  production  rate  of  propionate  increased  in  AII-­30  and  ST-­70  groups.  The   families  Veillonellaceae  and  Prevotellaceae  are  linked  to  propionate  production  (De   Menezes  et  al.,  2011,  Poudel  et  al.,  2019)  and  an  increased  abundance  of  both  families   is  visible  during  acidosis  period  in  certain  treatment  groups.  In  an  in  vivo  SARA  trial   Kmicikewycz   and   Heinrichs   (2014)   intended   to   increase   the   growth   of   these   starch   fermenting  bacteria  by  feeding  a  high  ground  wheat  ratio.  However,  the  high  starch   feeding  did  not  affect  the  propionate  production.  Besides  producing  acetate  and  little   amounts  of  butyrate,  Selenomonas  ruminantium  is  also  linked  to  propionate  production   by   succinate   decarboxylation   (Russell   and   Baldwin,   1979).   Throughout   all   three   experimental   periods,   the   butyrate   production   remained   stable,   however,   due   to   a   decreasing   acetate   production,   the   molar   proportion   of   butyrate   increased   during   acidosis  challenge.  The  Gram-­positive  bacteria  Butyrivibrio  (especially  B.  hungatei  and   B.   fibrisolvens)   and   Pseudobutyrivibrio,   both   belonging   to   the   family   of   Lachnospiraceae,   as   much   as  Clostridium   proteoclasticum   are   known   to   produce   butyrate  during  high  grain  diet  feeding  (Mrazek  et  al.,  2006,  Paillard  et  al.,  2007).  In   the  presented  work,  the  genus  Butyrivibrio  sp.  was  less  abundant  during  the  low  pH  

phase.  The  family  of  Lachnospiraceae  decreased  during  acidosis  period  in  the  solid   fraction,  however,  increased  during  the  second  control  period.  

In   the   present   work,   the   ammonia-­N   concentations   were   not   influenced   by   low   pH   values.   This   appears   to   be   contradictory   to  in   vivo   and   other  in   vitro   observations.  

Several  in   vitro   experiments   reported   decreasing   ammonia   concentrations   during   SARA  periods  (Mickdam  et  al.,  2016,  Eger  et  al.,  2017).  Erfle  et  al.  (1982)  reported  a   diminished  ammonia  concentration  in  vitro,  when  pH  levels  decreased  below  pH  6.0.  

Authors  conclude  that  a  washout  of  proteolytic  bacteria  below  pH  5.5  and  a  diminished   deaminase  activity  below  pH  6.0  were  the  reasons  for  the  low  NH3-­N  concentrations.  

This  is  in  line  with  an  in  vivo  trial  by  Lana  et  al.  (1998).  The  ammonia  concentration   declined  when  the  ruminal  pH  decreased  below  pH  5.7  in  animals  adjusted  to  a  high   forage  feeding.  The  ammonia  concentration  in  cattle  adjusted  to  a  90%  concentrate   feeding   was   generally   lower   compared   to   the   high   forage   fed   animals   (Lana   et   al.,   1998).  These  results  suggest,  that  the  ammonia  production  is  influenced  by  lower  pH   and  the  substrate  provided  (Bach  et  al.,  2005).    

4.2.2   The  roughage-­to-­concentrate  ratio  influences  the  ruminal  fermentation