Nitrogen-fixing Bacteria

Eric, Paula, Joy, Chi-Chien

Introduction

Only prokaryotes are able to fix nitrogen. Fixing nitrogen is distributed among

oxygenic and anoxygenic phototrophic bacteria, chemoautotrophic and

chemoheterotrophic bacteria, among aerobes and anaerobic bacteria - in short all major

physiological groups. Many bacteria have not been tested for there ability to fix nitrogen.

The majority of aerobic nitrogen-fixing bacteria are microaerophyllic in the sense that they

can not grow above 0.02 bar of oxygen and still have there nitrogen -fixing ability.

The great problem of nitrogen-fixation is the nitrogenase sensitivity to oxygen.

How do several aerobic bacteria, such as Azotobacter and cyanobacteria, manage to

tolerate oxygen partial pressure of the air? The nitrogen-fix bacteria also reduce protons.

For that reason they have a hydrogenase. Recently a close relationship between hydrogen-

oxidizing bacteria and nitrogen-fixing bacteria have been seen, Xanthobacter

autotrophiens and Alcaligenes latus, which are hydrogen-oxidizing bacteria are able to fix

nitrogen.

Materials and Methods

Purple non-sulfur diazotrophic bacteria were grown on the medium described

in the handout on isolating PNSB. School Street Marsh water sample were filtered and

plated on agar medium that lacked ammonium. The medium was put in Gas-Pak jars and

incubated in the light. The class has 12 more PNSB that were tested for the ability to

grow with out ammonium add to the medium.

Aerobic diazotropbs were isolated on Burk’s medium: 0.2 gIL KH2PO4,0.8 gIL

K2HPO4,0.2 gIL MgSO4,0.09 gIL CaCI2, 1.0 ml Na2MoO4,1.0 ml FeSO4,20.0 gIL

sucrose, 15.0 gIL Bacto Agar wash 3 times, pH 7.2 (0.25 mg/mLNa2MoO4,5.0 mg/nil

FeSO4). School Street Marsh and Bell Tower Park ditch water was inoculated into 100

mL Burlà medium without agar containing benzoate, sucrose, and mannitol as carbon

source. School Street Marsh samples were filter and plated on Burk’s medium with Mo

and without Mo.

Clostridium diazotrophs were looked for. The medium that was used is as

follows: 0.8 gILK2HPO4,0.2 gIL KH2PO4,10 mgIL yeast extract, 20 gIL sucrose, 0.2 gIL

Mg504,0.2 gIL NaCI, and 1.0 ml 5L12(trace element). Three 25 ml of medium was

inoculated with soil from outside LEOB, one was pasteurization and then incubated in the

anaerobic chamber, the second one was incubated in the anaerobic chamber, and the third

one was left outside the anaerobic chamber at room temp. When they cultures looked

turbid they were streak on plates and isolated colonies were picked.

Acetylene reduction assay was done by taking a 100 mL culture with 35 mL of

gas space in the bottle. Then we withdrew 3.5 mL of the gas space and then immediately

injected 3.5 mL of acetylene into the gas space. Then we looked for ethylene production

on the gas chromatograph.

Results

The purple non-sulfur bacteria from the class there were 12 pure colonies of these

there were 9 colonies that grew in the absence of anitnonium. One clearly failed to grow

and two grew only weakly. Therefore, 75% of the PNSB previously isolated were able to

grow without added amnionium. We filtered 0.1, 1.0, and 10 niL of School Street Marsh

water on PNSB medium that lacked ainmonium and were incubated in the light. Colonies

were streaked until apparent purity was obtained. We have isolated 8 cultures from the

School Street Marsh of these all of them grew in absence of atumonium. Of these 8

colonies all of them grew in the dark in the absence of ammonium.

We did an acetylene assay on one of the colonies that were isolated, Purple B. At

time 0 there was 698, 110 area for the acetylene peak and 0 area for the ethylene peak. At

time 2 h there was 671, 077 area for acetylene peak and 18, 885 area for the ethylene

peak. At time 4 h there was 615, 447 area for acetylene peak and 99, 606 for the ethylene

peak. At 21 h there was 545, 770 area for acetylene peak and 192, 871 area for the

ethylene peak. At 72 h there was 54, 794 area for acetylene peak and 658, 012 area for

the ethylene peak.

Aerobic diazotroph could not grow in the liquid medium either from School Street

Marsh or Bell Tower Park ditch. There was a single bacteria that grew when we filtered

on the Mo+/Mo- Burk’s agar, the Mo- did not grow as well as the Mo+. This strain was a

FRSC

gram negative slight curve rod and it was catalase +. When it was tried to grow in liquid

medium it did not so, we could not do an acetylene assay on the aerobic diazotroph.

Isolating a Clostridium species that was a diazotroph. Microscopically, from the

three 25 mL flasks the one that was pasteurized had mostly endospore formers, the second

one was half was endospore formers, and the third one only had a few endospore formers.

The liquid cultures for the one that was pasteurized was streaked onto plates. Two

colonies were isolated from the plated and restreak until pure culture was obtain. A non

endospore former was obtain and a endospore former was obtain. The non-endospore

former was facultative and the endospore former was also facultative. They could grow in

the absence of ammonium though. However, they could not be isolated in liquid medium,

so we could not do an acetylene assays.

Discussion

The purple non-sulfur bacteria that were isolated in the class, which were not

required to grow in the absence of ammonium, 75% of these colonies could fix nitrogen.

Of the 8 that we found all of them were able to fix nitrogen, because this was one of the

enrichment techniques we used. They could grow in anoxygenic phototroph without

nitrogen added to the medium, they could grow heterotrophically without light without

added nitrogen. We checked this by doing the acetylene assays and they could reduce

acetylene to ethylene. This is a quick way of seeing whether your organism fix-nitrogen.

EPSC

.A •c

The aerobic diazotroph we only had one colony that grew up, and it seemed that it

need molybdenum for growth. Either it switched from the molybdenum nitrogenase to the

vandenate nitrogenase or the molybdenum could not be extracted from the medium,

because we still got a little growth without the rnolyddenum. We could not get any

cultures to grow in a liquid medium. We might try a soil sample in addition the water.

Mother possibility is that the culture did not adequately mix when placed on the shaking

water bath.

We have not gotten a Clostridium species. We have gotten two other organism,

but they were both facultative anaerobes. The anaerobic chamber still had oxygen in

them. Maybe this had something do with isolating a strict anaerobe.