mcb 150 exer8 post lab

7/31/2019 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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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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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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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

mcb 150 exer8 post lab

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