Transcript The rumen bacteria - Prof. G. R. Ghorbani l Ghorbani.iut.ac.ir

The rumen
The rumen
A fermentation vat converting
plant materials to VFA’s, CH4,
CO2 NH3 and microbial cells
38 to 42oC
pH normally 5.5 to 6.5
maintained by
phosphate and bicarbonate ions in
saliva
rapid absorption of VFA’s and
ammonia
Bacteria growth is limited
mainly by availability of energy
yielding substrates and factors
such as pH
Rumen
Characteristics
Rumen DM is 10-18%
Osmolarity is usually <400
mOsmol/kg
Oxidation-reduc pot is -0.35V
Gas phase 65% CO2
27% CH4
7% N2
0.6% O2
0.2% H2
0.01% H2S
Rumen
Characteristics
Volatile fatty acids
66 mM Ac
23 mM Pr
10 mM But
2 mM higher c
Why rumen envir.
reduced
 Oxygen taken in with the food
and water is rapidly
metabolized by aerobic
organism
 CO2 and CH4 displces O2 from
the fluid
 Rumen bact produces
reducing substances such as
sulfide
Establishment of rumen
microorganisms
Days after birth
0
5
Facultative Celluloly
anaerobes tic
populati
Microaeroph
on
ilic
15
20
25
10
Fungi
Caecomyces
Piromyces
Neocallimastix
50
Protozoa, Mycoplasma
EntodiniaDiplodinia
Holotrichs
Rumen Microbial
Symbionts
PROVIDE
Energy
VFA can provide up to 80% of the
energy needs
Protein
microbes convert NPN into
high quality protein
Vitamins
synthesis of B-complex and
K vitamins
Detoxifying functions
Groups of Bacteria in the
Rumen
1. Free-living in the liquid phase
2. Loosely associated with feed
particles
3. Firmly adhered to feed particles
4. Associated with rumen epithelium
5. Attached to surface of protozoa
and fungi
Bacteria Associated with Feed
Particles
Groups 2 and 3
75% of bacterial population in rumen
90% of endoglucanase and xylanase
activity
70% of amylase activity
75% or protease activity
Substrate specificity
The basis for role assignation
for bacteria; tremendous
variation
Bulk of knowledge based on
studies with cultivated species
poor representation ?
evolution during propagation ?
Polymer utilisers and utilisers of
hydrolysis and fermentation
products
specialists
generalists
Bacterial
forms
rods
cocci
Spirochete
filamentous
budding and appendaged
Cocci may be found in different
arrangements
single
pairs
groups
chains
Fibrobacter
succinogenes
Gram - none motile rods but
can become coccoid or oval on
culture
Fibrobacter
succinogenes
Reportedly most widespread
cellulolytic bacteria; about 5 %
of all rumen isolates
Most extensive lignocellulosic
degradation in vitro; ferment
cellobiose and glucose
Do not hydrolyse xylan and
make limited use of pentoses
liberated from fiber digestion
Fibrobacter
succinogenes
Some strains hydrolyse starch,
pectin and lactose
Not proteolytic; utilises NH3,
amino acids and di-peptides as
nitrogen (N) sources
Major fermentation products are
A and S; but may also produce F,
P and isovalerate (iV)
H2 and CO2 not produced
Sensitive to low pH; relatively
resistant to antibiotics
Ruminococcus
species
Gram + non motile cocci
2 cellulolytics: R. albus and R.
flavefaciens
Most active degraders of plant
fiber
Ruminococcus
species
Both exhibit a glycocalyx
(attachment) and cell surface
protrubances (enzyme
complexes ?)
R. albus more numerous but
not all strains are cellulolytic;
produces yellow pigment when
grown on cellulose
Both degrade xylan and
ferment cellobiose but only R.
albus ferments glucose
Ruminococcus
species
Not proteolytic; require NH3 for
growth
Major fermentation products
are A, and A, S for R. albus
and R. flavefaciens
respectively
H2 and CO2 also produced
Sensitive to monensin and low
pH
Third species, R. Bromii an
important starch digester
Ruminobacter
amylophilus
Oval to long Gram - rods
Dominant starch digesters
hence, prevalent on grain diets
Ruminobacter
amylophilus
Specialists; utilise mainly
starch and maltose
Intracellular starch hydrolysis
Highly proteolytic
Major fermentation products
A, S, F (L)
H2 and CO2 not produced
Streptococcus
bovis
Gram + non motile oval to
coccoid cells
Strict anaerobe and aerotolerant strains; prevalent on
grain diets
Most rapidly acting degraders
of starch; ferment a wide
range of hydrolysis products of
plant polymers
Major fermentation product is
L, (F,A)
Co2 produced
Streptococcus
bovis
Capable of growing at low pH
(<5.0)
Play a major role in
development of lactic acidosis
Proteolytic
Succinomonas
amylolytic
Gram + straight rods or
coccobacilli motile by single
flagellum
Specialists. Predominantly
starch digesters; ferment
glucose and maltose
Fermentation products mainly
S, (A, P)
H2 and CO2 not produced
Butyrivibrio
fibrisolvens
Gram - B producing bacteria
Genetically very diverse;
taxonomy undergoing redefinition
Occur singly, in pairs or chains;
motile by means of polar
flagellum; posses capsular
material
Generalists. Important role in
degradation of starch, xylan and
pectin hence dominant on diets
ranging from grain to alfalfa hay
Butyrivibrio
fibrisolvens
Proteolytic
Cellulolytic isolates; but ability
not present in lab cultures
Major fermentation products:
B, F and A (L and S)
H2 and CO2 also produced
Lachnospira
multipara
Pectin degrading Gram +
curved rods
Motile by means of 1 lateral
flagellum
Capsular material similar in
composition to B. fibrisolvens
High concentrations on forage
legumes
Major fermentation products:
F, A and L.
H2 and CO2 also produced
Proteolytic
Prevotella species
Gram + rods or cocci;
numerous
4 separate species now
identified
Prevotella species
Generalists. Degrade starch,
xylan and pectins but not
cellulose
Proteolytic and play critical
role in uptake and
fermentation of peptides
Produce A, F, S, P, isobutyrate
(iB), other minor FA’s too
Succinivibrio
dextrinosolvens
Gram - helically twisted rods;
polar flagellum
Succinivibrio
dextrinosolvens
Hydrolyse dextrin (starch
diets) and grass levans
Some strains ferment end
products of plant cell wall
degradation e.g. cellobiose
Major fermentation products
are A, S and F (L)
Anaerovibrio
lipolyptica
Gram - rods motile normally by
single polar flagellum; some
isolates have multiple flagella
Specialists. 3 key properties
hydrolyse lipids, utilise lactate
and ferment fructose
Fermentation products: P, S, A
(L)
H2 and CO2 also produced
Utilisers of
hydrolysis products
Selenomonas
ruminantium
Distinctive Gram - curved rods;
linear array of upto 16 flagella
on concave side
Selenomonas
ruminantium
Prevalent on cereal grain diets
Proteolytic
Utilises mainly sugars; some
strains hydrolyse starch; most
strains are unable to degrade
pectins and xylans
Some strain utilise lactate for
growth
Fermentation products: either L
or P and A when grown on high
and low concentrations of
glucose, respectively
H2 and CO2 also produced
Megasphaera
elsdenii
Gram - non motile cocci
occurring in pairs and chains
of upto 20 cells
Young animals and animals of
high grain diets
Utilises wide range of
degradation products such as
sugars but not polymers
Wide range of fermentation
products depending on
substrate
Megasphaera
elsdenii
Ferments L to mainly B, P, iB, V
Ferments glucose to mainly
caproate and F with some A, P, B
and V
H2 and CO2 also produced
Important roles include
production of branched chain
fatty acids from amino acids
Methanogenesis
Exclusively in anaerobic
environments; 5 to 110oC; fresh
water to salt water;
Terminal step in carbon flow
Methanogens: archaea not
bacteria
Strict anaerobes: most difficult
rumen microbes to culture in
vitro
O2 conc of 10-56 required for CH4
production and growth
require simple molecules for
growth
Rumen
methanogens
Convert 800 L H2 to 200 L CH4
in a 500 kg dairy cow in a day
9 to 25 % associated with
protozoa
Maintain low H2 partial
pressures in rumen and thus
allow for reduced cofactors in
metabolic pathways (NADH)
to be oxidised (NAD)
VFA’s are the main products of
this process
Rumen
methanogens
Methanobrevibacter
ruminantium*
Methanobacterium formicicum
and Methanomicrobium mobile
(rods)
CH4 from H2, CO2 and formate
Methanosarcina barkeri (cocci)
CH4 from H2, CO2 (slow growth)
but mainly from acetate,
methanol and methylamines
Isolation of rumen
bacteria
Usually total and cellulolytic
bacteria
amylolytic, xylanolytic and
saccharolytic e.t.c.
Sampling
Cannulated animals
Multi-site sampling
Minimize headspace
Homogenise under CO2; filter
through cheese cloth into prewarmed (ca 39oC) thermos
Viable cell
count
1
ml
1.dilute
sample
9 ml
107
cell
no/ml100 10
1000
2. plate out 0.1
ml
106
105 104
Isolation of bacteria
Solid associated microorganisms
may be detached using methyl
cellulose (or tween-80 followed
by chilling for 6 - 8 h)
Dilute sample in a 10 fold
dilution series using an
anaerobic diluent
Role tube method employed
for all but cellulolytic
Anaerobic and sterile
procedures
Enumeration of
bacteria
For cellulolytics: MPN procedure.
• 1 ml from 10-6 to 10-8 dilutions
inoculated in triplicate into 4 ml
anaerobic growth media (39oC) in
Hungate tubes, containing a strip of
filter paper as sole C source. Incubate
at 39oC for up to 2 weeks
• Numbers determined statistically
based on numbers of tubes containing
digested filter paper at each dilution
For other groups,
• Same procedure but inoculate (10-6 to
10-9) into media containing 1.8 %
agar(47oC) and either a mixture of
energy sources for total counts, or
just the specific substrate for each sub
group. Spin tubes in ice slurry to
solidify agar
• Incubate at 39oC for 2 to 3 days and
count colonies (/ml rumen fluid)
1. Biochemical requirements
for growth
Culture media
Water + nutrients
energy source;organic,
inorganic or light
carbon, nitrogen source
Agar can be added to make the
medium semi-solid and poured into
Petri dishes
2. Physical and environmental
requirements for growth
• oxygen concentration
• pH (hydrogen ion
concentration)
• temperature
Growth media
Rumen fluid containing
• Clarified autoclaved rumen fluid (10 to
20 %)
• Mineral solution 1: KH2PO4
• Mineral solution 2: NaCl, (NH4)2SO4,
CaCl2, MgSO4 and microelements
• VFA and Vitamin solutions
• Resazurin
• Energy substrate(s) as required
• Casein hydrolysate (amino acids and
peptides)
• pH adjusted to 6.5
Boil; replace headspace with
stream of CO2
• Reducing agent and bicarbonate
• Dispense in to tubes, seal and
autoclave
Growth media
Chemically defined media
No rumen fluid; nutrient
requirements usually met by
rumen fluid must be supplied e.g
phenylpropanoic acid for R.
albus and 1, 4-napthoquinone
for S. dextrinosolvens.
Anaerobic glove box
Permits growth of anaerobes on
Petri dishes, hence the use of
techniques such as
• replica plating
• rapid identification systems such as
strip tests
avoids exposure of bacteria to
molten agar (47oC)
Sufficient CO2 in atmosphere to
maintain medium pH
for nutrition of microbes requiring
CO2
95 % CO2/ 5% H2 ok for most
rumen bacteria; for
methanogens, 80% H2/20% CO2
An anaerobic glove
box
Maintenance of
cultures
Storage
at 4oC can retain viability for up to
1 month (storage usually for up to
1 week)
of 12 to 24 h cultures in 20 %
glycerol for up to 1 year
under liquid nitrogen (-196oC);
followed by rapid thawing in water
at 32 to 35oC. Very successful.
Freeze drying. Reports of viability
for up to 5 years
Conversion of
carbohydrates to
pyruvate
Methanogenesis
Three major catabolic pathways
CO2 reduction*
• CO2 and H2 to form CH4; electrons
mainly from free H2 but also formate
Methyltrophic pathway
• reduce CH3 groups from compounds
methanol, trimethylamine
Aceticlastic pathway
• Split acetic acid and reduce CH3 to CH4