autotrophiens and Alcaligenes latus, which are hydrogen-oxidizing bacteria are able to fix Nitrogen-fixing Bacteria Eric, Paula, Joy, Chi-Chien

Transcription

1 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 hydrogenoxidizing 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

2 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. K2HPO Aerobic diazotropbs were isolated on Burk s medium: 0.2 gil KH 4, 0.2 gil MgSO 4, 0.09 gil CaCI 2MoO 2, 1.0 mlna 4, 1.0 ml FeSO 2PO4, 0.8 gil 4, 20.0 gil 4, 5.0 mg/nil sucrose, 15.0 gil Bacto Agar wash 3 times, ph 7.2 (0.25 mg/mlna 2MoO FeSO 4). 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 gil K2HPO Mg50 4, 0.2 gilkh 2PO4, 10 mgil yeast extract, 20 gil sucrose, 0.2 gil 4, 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.

3 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

4 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 ofthe 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

5 .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.