Mcb 150 Exer8 Post Lab
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Transcript of Mcb 150 Exer8 Post Lab
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GROUP 2WINDY LOU BAGAY
PRECIOUS DELA CRUZ
CYRENE D. ESTRELLANA
REYJONDEL MARASIGAN
CARL DAVE S. MENDOZAMCB 150 C-3L
Exercise 8:
DEMOSTRATION OF A BIOGEOCHEMICALCYCLE
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BIOGEOCHEMICAL CYCLE
Oxidation and reduction of substances that
results in the cycling of elements within and
between different environments
Carried out by microorgsnisms
cycles and their many feedback loops are
essential for plant agriculture and the overall
health of sustainable planet life
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BIOGEOCHEMICAL CYCLE
Carbon cycle
Nitrogen Cycle
Sulfur Cycle Iron Cycle
Phosphorus, Calcium, and Silica Cycles
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NITROGEN CYCLE
involves the reduction and oxidation
reactions, change in form and location of
nitrogen on earth
Highy dependent on the action of
microorganisms
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NITROGEN CYCLE
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OBJECTIVES
To demonstrate steps in nitrogen cycle using
control organisms and soil sample;
To describe the enzymatic activities of
organisms involved in the nitrogen cycle;
To be able to define the importance of
microorganisms in biogeochemical cycles.
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AMMONIFICATION
Decomposition of organic nitrogen such as
amino acids and nucleotides to ammonia
Ammonia (NH3
)exist as ammonium ion
(NH4+ ) at neutral pH
In alkaline soils, ammonia is lost by
vaporization because it is volatile
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AMMONIFICATION
Ammonifying bacteria:
Bacillus
Clostridium
Proteus
Pseudomonas
Streptomycin
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AMMONIFICATION (EXERCISE)
Peptone broth
Provides substrates for ammonification
Two test:Qualitative
Quantitative
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AMMONIFICATION (EXERCISE)
Qualitative test
Nesslers Reagent (potassium tetraiodomercurate)
Positive reaction= yellow
Reaction:
NH4+ + 2[HgI4]
-2 + 4OH- HgOHg(NH2)I + 7I-
(yellow)+ 3H2O
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AMMONIFICATION (EXERCISE)
Qualitative test
0.1N sulfuric acid (titrant) and Methyl red
(indicator)
Methyl red: red=pH below 4.4
yellow=pH above 6.0
high volume of acid used= high amount of NH3
(basic)
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AMMONIFICATION (EXERCISE)
mg of NH3 in 100 ml
=T value x N of acid x equiv.wt. of NH3 x 100
vol of sample
T value= ml of acid used in sample-ml of acid used on control
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Samples/Cultures Nesslerstest (+/-)
mg of NH3/100 ml
B. cereus - 112P. fluorescens + 22.4P. vulgaris + 11.2Non-acid, non-
waterlogged soil+ 700
Water logged soil
Acid soilControl - 0
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NITRIFICATION
Process by which ammonium or ammonia is
oxidized into nitrite (NO2-), which is further
oxidized to nitrate (NO3-)
Occurs only under aerobic condition
Carried out by nitrifiers (strict aerobe)
Nitrite formation
Nitrate formation
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NITRIFICATION
Factors affecting the rate of nitrification
TemperatureEnzyme action
Oxygen concentrationAerobic condition only
pHReactions fastest when pH is from 7.5 to 8.5
Inhibiting substancesMetals
Nitrapyrin (inhibits the first step in nitrificatio)
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NITRITE FORMATION (NH4+ NO2
- )
By nitrosofying bacteria
Nitrosomonas, Nitrosovibrio, Nitrosococcus, Nitrosolobus
3 sub-processes
NH3
is oxidized into hydorxyl amine (NH2OH) by the
enzyme mono-oxygenase
NH3 + O2 + 2H+ + 2e- NH2OH + H2O
NH2OH is oxidized into NO2-
NH2OH + H2O NO2- + 5H+ + 4e-
Electrons and oxygen and free hydrogen ions areconverted to water
O2 + 2H+ + 2e- H2O
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NITRITE FORMATION (NH4+ NO2
- )
Over all reaction
NH3 + 1.5O2NO2- + H+ + H2O
Acidifies the environment
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NITRITE FORMATION (EXERCISE)
Nitrite formation medium (ammonium sulfate
broth)
Test for presence of NO2-: Trommsdorfs
solution + 1: 3 sulfuric acid-water solution
(+)= intense blue-black color
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NITRATE FORMATION (NO2- NO3
- )
By nitrifying bacteria Nitrobacter, Nitrospira, Nitrococcus
Enzyme: nitrite oxidoreductase (NOR)
2 steps: NO2- is oxidized into NO3- by NORNO2
- + H2O NO3- + 2H + + 2e-
Remaining oxygen, electrons and protons assembles intowater
O2 + 2H+
+ 2e-
H2O Over all reaction
NO2- + O2 NO3
-
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NITRATE FORMATION (EXERCISE)
Nitrate formation medium (nitrite broth)
Test for presence of NO3- : diphenylamine
reagent and conc. sulfuric acid
(+)= dark blue-black color
Must be done only after test for nitrites turn
negative because diphenylamine gives a
positive for both nitrites and nitrates
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Soil sample Nitrite
FormationNitrate Formation
Trommsdorfs
solutionDiphenylamine
reagentNon-acid, non-
waterlogged soil - + (still + after 1week)
N/AWaterlogged soil
+ - +Acid soilControl
- - -
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NITRATE FORMATION (EXERCISE)
(-) for NO3- :
Longer incubation period is needed for full
conversion of NO2- to NO3
-
Reduced growth rate of nitrifying bacteria due toacid produced during nitrification (nitrification
stops at a pH below 6.0)
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DENITRIFICATION
Biological reduction of nitrate to gaseous
nitrogen compounds by facultative
heterotrophic bacteria or denitrifiers
Bacillus, Paracoccus, Pseudomonas,Alcaligenes, Azospirillum, Rhizobium,Rhodopsuedomonas, Propionibacterium
occurs when oxygen levels are depleted(under strict anaerobic conditions or reduced
oxygen tension)
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DENITRIFICATION
Dissimilatory nitrate reduction
Or nitrate respiration
When NO3- ions act as terminal electron
acceptors on the absence of oxygen
NO3- is converted into variety of reduced
products
Enzyme: dissimilatory nitrate reductase Inhibited by oxygen
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DENITRIFICATION (EXERCISE)
Nitrate broth KNO3 or NaNO3 as NO3 source
with durham tubes
To detect gas production Filled tubes to total capacity and covered with
rubbedr stopper
To prevent entry of atmospheric gases especially
oxygen
Test for pH, gas production and presence ofNH3, NO2 and NO3
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Soil Sample pH Production of substancesAmmonia
(+/-)Nitrite
(+/-)Nitrate
(+/-)Gas
Non-acid,
non-
waterlogged9 + - - -
Waterlogged 8.5 + - - +AcidicP. aeruginosa 8.5 + - - +Negative
control 8 + - - -
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DENITRIFICATION (EXERCISE)
(-) for NH3 = may have been vaporized due
to alkaline condition
(-) for nitrate = may have been reduced to
other N gas compounds
(+) for nitrite = nitrate was utilized as electron
acceptor and converted to nitrite
(+) for gas = nitrite is converted to N2 or N2O
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NITROGEN FIXATION
Process where N2 gas is converted into NH3
by diazotrophic bacteria
Involves a sequence of reduction steps that
require major energy expenditures due to
stability of the dinitrogen triple bond
Enzyme: nitrogenase
Oxygen sensitive
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NITROGEN FIXATION
In aerobic nitrogen fixing bacteria,nitrogenase inactivation by oxygen wasprevented by
Rapid removal of oxygen by respirationBiofilm formation
Compartmentalization (heterocyst formation)
Conformational protection (complexing withspecific protein
Occurs in response to low concentration ofammonia
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NITROGEN FIXATION
Occurs in environments with abundant
carbon source and low nitrogen
Two types:
Symbiotic nitrogen fixation
Non symbiotic nitrogen fixation
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SYMBIOTIC NITROGEN FIXATION
Involves symbiosis of organisms
By marine diatoms, bivalves, plants with
nodules in their adventitious roots
Examples: Rhizobiumand Bradyrhizobium
Plant with root nodule: Mimosa pudica
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Characteristics ObservationLocation Upper part of the
root
Nature of
attachmentOn the side
Shape and size
of noduleOblong, ~1mm
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SYMBIOTIC NITROGEN FIXATION (EXERCISE)
Microscopic observation
directly form the nodule
grown in Yeast Mannitol Agar
used for the cultivation ofRhizobiumsp. and forstudying root nodulation
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SYMBIOTIC NITROGEN FIXATION (EXERCISE)
Mannitol - fermentable sugar alcohol source
Magnesium - provides cations essential for the growth
ofRhizobia
Yeast extract - amino acids, vitamin B complex and
accessory growth factors for Rhizobia; provides
oxidation-reduction potential of medium in the range
favorable for Rhizobia; serves as hydrogen donor in
respiratory process
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NON-SYMBIOTIC NITROGEN FIXATION
free-living nitrogen fixers
Azotobacter, Clostridium, cyanbacteria, purpleand green bacteria
In the exercise:N-free glucose medium
- Observed for biofilm formation
N-free Iron-free Glucose agar
-presence of pigment
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NON-SYMBIOTIC NITROGEN FIXATION
Biofilm formation: to retard oxygen diffusion
that can inactivate the nitrogenase enzyme
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Thank you!!!
END