Exercise 8: DEMOSTRATION OF A BIOGEOCHEMICAL CYCLE

GROUP 2 WINDY LOU BAGAY PRECIOUS DELA CRUZ CYRENE D. ESTRELLANA REYJONDEL MARASIGAN CARL DAVE S. MENDOZA MCB 150 C-3L

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

BIOGEOCHEMICAL CYCLE
Carbon cycle Nitrogen Cycle Sulfur Cycle Iron Cycle Phosphorus, Calcium, and Silica Cycles

NITROGEN CYCLE
involves the reduction and oxidation reactions, change in form and location of nitrogen on earth Highy dependent on the action of microorganisms

NITROGEN CYCLE

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.

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

AMMONIFICATION

Ammonifying bacteria:
Bacillus
Clostridium Proteus

Pseudomonas
Streptomycin

AMMONIFICATION (EXERCISE)
Peptone

broth
substrates for ammonification

Provides

Two

test:

Qualitative
Quantitative

AMMONIFICATION (EXERCISE)

Qualitative test
Nesslers

Reagent (potassium tetraiodomercurate) Positive reaction= yellow Reaction:

NH4+ + 2[HgI4]-2 + 4OH- HgOHg(NH2)I + 7I(yellow)+ 3H2O

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)

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

Samples/Cultures

Nesslers test (+/-)

+

mg of NH3/100 ml
112 22.4

B. cereus P. fluorescens

P. vulgaris
Non-acid, waterlogged soil Water logged soil non-

+
+

11.2
700

Acid soil
Control 0

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

NITRIFICATION

Factors affecting the rate of nitrification

Temperature
Enzyme

action

Oxygen pH

concentration
condition only
fastest when pH is from 7.5 to 8.5

Aerobic

Reactions

Inhibiting
Metals

substances
(inhibits the first step in nitrificatio)

Nitrapyrin

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 NO2NH2OH + H2O NO2- + 5H+ + 4e Electrons and oxygen and free hydrogen ions are converted to water O2 + 2H + + 2e- H2O

NITRITE FORMATION (NH4+ NO2- )

Over all reaction NH3 + 1.5O2NO2- + H+ + H2O Acidifies the environment

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

NITRATE FORMATION (NO2- NO3- )

By nitrifying bacteria

Nitrobacter, Nitrospira, Nitrococcus

Enzyme: nitrite oxidoreductase (NOR) 2 steps:

NO2- is oxidized into NO3- by NOR NO2- + H2O NO3- + 2H + + 2e Remaining oxygen, electrons and protons assembles into water O2 + 2H + + 2e- H2O

Over all reaction NO2- + O2 NO3

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

Soil sample

Nitrite Formation

Nitrate Formation Trommsdorfs solution Diphenylamine reagent

Non-acid, nonwaterlogged soil

+ (still + after 1 week) -

N/A

Waterlogged soil
Acid soil Control

NITRATE FORMATION (EXERCISE)

(-) for NO3- :

Longer

incubation period is needed for full conversion of NO2- to NO3 Reduced growth rate of nitrifying bacteria due to acid produced during nitrification (nitrification stops at a pH below 6.0)

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)

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

DENITRIFICATION (EXERCISE)

Nitrate broth

KNO3 or NaNO3 as NO3 source

To detect gas production

with durham tubes

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 of NH3, NO2 and NO3

Soil Sample

pH

Production of substances Ammonia Nitrite (+/-) (+/-) Nitrate Gas (+/-)

Non-acid, nonwaterlogged Waterlogged

Acidic

9
8.5

+
+

P. aeruginosa 8.5 Negative control

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

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

NITROGEN FIXATION

In aerobic nitrogen fixing bacteria, nitrogenase inactivation by oxygen was prevented by

Rapid

removal of oxygen by respiration Biofilm formation Compartmentalization (heterocyst formation) Conformational protection (complexing with specific protein

Occurs in response to low concentration of ammonia

NITROGEN FIXATION
Occurs in environments with abundant carbon source and low nitrogen Two types:

Symbiotic

nitrogen fixation Non symbiotic nitrogen fixation

SYMBIOTIC NITROGEN FIXATION

Involves symbiosis of organisms By marine diatoms, bivalves, plants with nodules in their adventitious roots Examples: Rhizobium and Bradyrhizobium Plant with root nodule: Mimosa pudica

Characteristics Observation Location Upper part of the root of On the side

Nature attachment Shape and size Oblong, ~1mm of nodule

SYMBIOTIC NITROGEN FIXATION (EXERCISE)

Microscopic observation
directly

form the nodule grown in Yeast Mannitol Agar

used

for the cultivation of Rhizobium sp. and for studying root nodulation

SYMBIOTIC NITROGEN FIXATION (EXERCISE)

Mannitol

- fermentable sugar alcohol source Magnesium - provides cations essential for the growth of Rhizobia 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

NON-SYMBIOTIC NITROGEN FIXATION

free-living nitrogen fixers

Azotobacter,

Clostridium, cyanbacteria, purple and green bacteria

In the exercise:
N-free

glucose medium - Observed for biofilm formation N-free Iron-free Glucose agar -presence of pigment

NON-SYMBIOTIC NITROGEN FIXATION

Biofilm formation: to retard oxygen diffusion that can inactivate the nitrogenase enzyme

Thank you!!!

END