Lab Project Two Symbiotic Nitrogen Fixation
Enchrichment & N Cycle Legumes: root hairs secrete inducer molecules
Symbionts: soil Rhizobium, Proteobacteria
Separate/no fixation; together/leghemoglobin
Enrichment Techniques Bacteroids: misshapen Rhizobium in nodules
Goal - tease target microbes from population
Inoculum - mixed sample with target microbes Free-Living Aerobic Azotobacter
Many strategies thereafter - physical, chemical Proteobacteria - capsule-producing rods
Understand inoculum, strategies - ALL projects! Beijerink - first historic enrichment (Fig. 22.1)
Nitrogen Fixation Nitrification, Aerobic Lithotrophy
N2 fixed to NH3 and NH2 groups - COSTS ATP Two-step - ammonia then nitrite oxidation
ONLY by Prokaryotes - Bacteria and Archaea NH3 + O2 yields NO2 + H2O, Nitrosomonas
Conserved O2-intolerant Nitrogenase/Fe-Mo NO2 + O2 yields NO3, Nitrobacter
Understand Acetylene Reduction Assay Both are Proteobacteria; packed with lamellae
Some Nitrogen Fixer Diversity Anaerobic Nitrate Reduction
Cyanobacteria: colonial, heterocysts resist O2 Denitrifying (to N2) OR Nitrifying (to N-fixed)
Methanogens/Archaea: live in anoxic habitats NO3 + CH2O yields N2 + CO2, Pseudomonas
PJ - Rhizobium: symbiotic leghemoglobin NO3 + CH2O yields NO2, NH3 + CO2, Enterics
PJ - Azotobacter: fastest ETC uses O2
Azotobacter - Work In Table Then Individually
As a table, add 1 g dry N-poor soil (e.g. outside west entrance) to 75 ml Azotobacter broth in flask
Incubate loosely covered on countertop for about a week - remember, these are free-living aerobes
Follow-up: each person streaks onto 1 Azotobacter plate, watch for formation of slimy colonies
Notebook records – in-color drawings, describe texture, shape, target colonies on final plates
Azotobacter N-Free Media
Prepared as liquid and agar (15-20 g/L) plates, with agar
Solution A Solution B
added to Solution B. Combine 1A:1B after autoclaving.
K2HPO4 : 1.6 g MgSO4: 0.4 g
This high-sugar enrichment medium is designed to
KH2PO4: 0.4 g CaSO4: 0.2 g
enhance capsule production, a distinct Azotobacter trait.
FeSO4/7H2O: 0.006 g
This medium is also highly selective for N2-fixers because it
MoO3: 0.002 g
contains no nitrogen.
sucrose: 10 g
Nitrate Reduction - Work In Table Then Pairs
As a table, gather 1 g organic soil (e.g. by NSM pond) and dilute in 9 ml water blank tube
Working in pairs, inoculate 4 nitrate reduction tubes: 2X1 ml above soil, E. coli, Pseudomonas
Follow-up: after a week, complete all portions of the nitrate reduction test as described below
Notebook records – in-color drawings, describe and interpret testing at each stage
Nitrate Reduction Media Prepared as 7 ml light gold liquid tubes with Durham tubes. After growth, read in
Beef Extract: 3 g order: (1) Bubble? (yes = NO3 to N2); (2) Add 10 drops A & B. Red? (yes =
Peptone: 5 g NO2); (3) If not, add 10 grains Zn and wait 2-3 minutes. Red? Yes = NO3
KNO3: 1 g present/no NO3 reduction; No = NH3, NH2-compounds.
Rhizobium - Work In Pairs Then Individually
Working in pairs, obtain 2 root nodules, lightly wash off dirt and surface treat 2 minutes, comparing…
1 nodule with bleach disinfectant; 1 nodule with alcohol antiseptic – KEEP TRACK which is which
After treatment, separately place each nodule in 1 drop sterile water - crush with sterilized forceps
Each person streaks (USE PATTERN) loop disinfected nodule crush onto Rhizobium plate
Each person streaks (USE PATTER) loop antiseptic-treated nodule crush onto Rhizobium plate
Follow-up: incubate plates on countertop about 1 week before looking for slimy colonies
Notebook records – in-color drawings, fully describe texture, shape, counts of final plates
Mannitol : 10 g
Prepared as agar plates (15-20 g/L). This high-sugar medium is
Yeast Extract: 1.0 g
designed to enhance capsule production, a distinctive
MgSO4/7H2O: 0.2 g
Rhizobium trait. These plates are not particularly selective and
NaCl: 0.2 g
must be used in conjunction with surface-sterilization for
K2HPO4: 0.5 g
FeCl3: 0.005 g
LAB MATERIALS TURN-IN
Due dates depend on enrichment timing, which can vary.
20 pts. Informal Notebook records: make sure all guidelines in syllabus and above are followed.
2.5 pts. INDIVIDUAL TURN-IN: Each person turns in his/her final Azotobacter plate, labeling an
Azotobacter colony. It will be graded on labeling, pattern, Azotobacter isolation/ID.
2.5 pts. INDIVIDUAL TURN-IN: Each person turns in his/her one best Rhizobium plate, labeling a
Rhizobium colony and indicating the surface treatment. It will be graded as above.
FORMAL SCIENTIFIC REPORT - ADVANCED METHODS (20 pts)
Each person will type out answers and turn in hard-copy Word reports.
Nitrosomonas and Nitrobacter are challenging to study using culture-based methods. For this
assignment, you will read and answer questions about research on these important bacteria using
advanced methods - FISH and microelectrodes, both applied directly to a living community. This
paper is linked to the class website and you will need to view it in color.
Paper: Schramm, et al., "Structure and Function of a Nitrifying Biofilm as Determined by In Situ
Hybridization and the Use of Microelectrodes," in Applied and Env. Microbiology, 1996, p. 4641-47.
Reading Comprehension Questions:
5 pts. Before you read the research paper, consult your textbook and - in your own words - explain
what FISH (including what the acronym stands for) and microelectrodes are, how they work, and what
kind of data they produce. You should spend a paragraph explaining each method.
4 pts. In terms of the research paper, what community did the scientists analyze - be careful: there
are two levels to this answer? What features of it make it a good place to study the target bacterial
groups? Would you call this a "natural" community - why and why not? You should spend a short
paragraph answering this question.
4 pts. In terms of FISH methods in the paper, please address the following in a short paragraph:
a. What are each of the following probes specific for: EUB338, NEU23a, NIT2, and NIT3?
b. Which table and/or figure represents FISH data? Explain colors in all images.
c. What is the most important take-home message the scientists discuss about the FISH data?
3 pts. In terms of paper microelectrode methods, please address the following in a short paragraph:
a. What did the scientists measure using microelectrodes?
b. Which table and/or figure represents microelectrode data? Explain experimental vs. control.
4 pts. Using the ASM (American Society for Microbiology) Journal website (there are search engines
within each journal section), locate another research article (must be a primary research paper – not
a review) that utilizes either FISH or microelectrodes. Download and print the cover of this paper and
staple it to your report. The cover must contain the title, author list, and abstract. NOTE: all ASM
articles are free after a year so make sure you choose something at least a year old.
ADDITIONAL ON-YOUR-OWN PROBLEMS
Be able to do these for the exam and/or quiz over this lab.
Would your Azotobacter colonies (as they exist on your plates) be positive using the acetylene
reduction assay? Would your Rhizobium colonies (as they exist on your plates) be positive?
Would you describe Azotobacter media as selective – why/why not? As differential – why/why not
and, if so, how/for what? Would you describe Rhizobium media as selective – why/why not? As
differential – why/why not and, if so, how/for what?
In the Azotobacter activity, what was the key strategy used to separate this target from the masses
and name a specific cell structure you saw. Answer the same question for Rhizobium.
Match to reactions below: Nitrobacter, Azotobacter, Nitrosomonas, Pseudomonas, Enterics, or NBP
(not biologically possible).
_____________________ N2 to NH3 _____________________ NH3 to NO3
_____________________ NO3 to N2 _____________________ NO3 to NH3
_____________________ NH3 to NO2 _____________________ NO2 to NO3
Draw N cycle from lab, labeling with above microbes. This would be graded ALL OR NOTHING.
Sample Multiple Choice
a. is symbiotically co-produced by Rhizobium and the its host legume
b. is made by some organisms in all three domains of life
c. activity can be tested for using the nitrate reduction assay
d. would have been active on Azotobacter plates, but not Rhizobium
e. more than one answer above is true
In nitrate reduction tube, denitrification was shown by
a. a bubble in the inner tube
b. no color change following A/B and zinc
c. red color change following A/B and zinc
d. no color change following A/B
e. red color change following A/B
Which solves the "nitrogenase-O2 problem" by most rapidly consuming O2 during respiration?
e. more than one answer above is true