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Old 12-31-2013, 03:05 PM   #181
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To be honest I'm not sure how to start backing all this up. What I read remains in my head. I have bookmarked a number of links though. What we would be best doing is finding somewhere we can compile all the we have learned so far and come up with some instructions for the experiments going forward.
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Old 12-31-2013, 03:20 PM   #182
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Google docs maybe? I don't think it has any country restrictions for us to both access/edit a public document. I'll look into it.

For now I'm just copying my stuff into a spreadsheet, but it makes even more sense if I put it somewhere you can see it too.
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Old 12-31-2013, 05:20 PM   #183
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My bucket cycle is approaching finished. The ammonia oxidizing bacteria are definitely "done" growing (as in, I don't need any more). They are clearing out 4ppm ammonia in 24 hours (or less, for all I know)
Now as soon as the nitrite oxidizing bacteria catch up it will be finished. Nitrates are so high I can't even read them because I don't know where it falls on the chart. Pretty red color though!
Levels 0/5/100+
Day 11
Never done any water changes (if it stalls now, we'll blame lack of phosphorus)

This is actually in line with how I was told this crazy dosing cycle should work. I'm quite excited.
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Old 12-31-2013, 07:57 PM   #184
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Fingers crossed!
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Old 12-31-2013, 08:58 PM   #185
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As to anyone who volunteered to do some cycling to post test data, I will PM you guys. I'll put up a google doc and you can either enter your test results to that or PM them to me.

I hope everyone is having a happy new year!
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Old 12-31-2013, 09:01 PM   #186
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Quote:
Originally Posted by threnjen View Post
As to anyone who volunteered to do some cycling to post test data, I will PM you guys. I'll put up a google doc and you can either enter your test results to that or PM them to me.

I hope everyone is having a happy new year!
So it took 11 days? Thats not bad at all. Still a week or so to go I reckon though

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Old 01-01-2014, 11:43 PM   #187
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Well with new year's I haven't been doing any reading!

I liked what you tried to do over in the newbie area, but it fell on deaf ears. I think it just goes over most people's heads, especially the newbies.

To be honest I think fish-in cycling needs to be at 1ppm ammonia or it will take forever. But I don't think you'll even agree with me, since you want a cycle test with .25
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Old 01-02-2014, 12:44 AM   #188
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I just read another study that directly contradicted your study about Nitrosomonas being affected by high nitrites. So, that's confusing. However my study is from 1998 and yours was 2002. They seem to directly contradict each other =|

Source: Loss of Ammonia Monooxygenase Activity in Nitrosomonas europaea upon Exposure to Nitrite

Nitrosomonas europaea, an obligate ammonia-oxidizing bacterium, lost an increasing amount of ammonia oxidation activity upon exposure to increasing concentrations of nitrite, the primary product of ammonia-oxidizing metabolism.”

“The loss of AMO activity via nitrite occurred under both aerobic and anaerobic conditions, and more activity was lost under alkaline than under acidic conditions except in the presence of large concentrations (20 mM) of nitrite. These results indicate that nitrite toxicity in N. europaea is mediated by a unique mechanism that is specific for AMO.”
High pH = more loss of activity

“ammonia oxidation is an acidogenic reaction. As the pH of the incubation medium decreases during ammonia oxidation, the NH3-NH4+ equilibrium is shifted away from NH3, the true substrate of AMO (23). Ammonia oxidation ceases at about pH 6, partially because of the reduced concentration of NH3
Just another place they talk about how the cycle reduces pH

“At the point of maximal cell growth in phosphate-buffered medium with an initial ammonium concentration of 50 mM, the concentration of nitrite is typically around 20 to 25 mM, and the rest of the ammonium remains unoxidized. In a previous study, we showed that this remaining NH3-NH4+ pool, or other substrates of AMO, had a specific protective effect on the AMO activity of N. europaea cells (22). In incubations where the ammonium was completely consumed, the cells lost up to 80% of their ammonia-oxidizing activity over a 24-h period (22). In the present paper, we show that this specific loss of AMO activity in cells of N. europaea is due to the toxicity of accumulated nitrite in the incubation medium.”
Underlined part = presence of ammonia protects the AMO from the high nitrites? Did I read that correctly?

“only a few reports describing the toxicity of nitrite in ammonia-oxidizing bacteria have been published, and the mechanism of toxicity remains unclear. In one study, nitrite toxicity in Nitrosomonas sp. occurred only at very high concentrations (greater than 30 mM) of nitrite, and this effect, as measured by the ability of the cells to consume O2, was greater during the lag phase than during the log phase of growth at several different pH values tested (16). Nitrite was also toxic for cells in the log phase of growth, but only at acidic pH values, and the loss of activity was reversible upon washing of the cells”

“Because batch cultures of N. europaea accumulate large concentrations of nitrite, greater than 20 mM, and reach a pH of about 6, it is surprising that the cells are not more susceptible to nitrite toxicity”

“The present study shows that nitrite can cause a specific loss of ammonia-oxidizing activity in N. europaea cells at lower concentrations than those previously considered (5 to 20 mM). The loss of activity is specific to AMO and occurs under both acidic and alkaline conditions. In the presence of substrates of AMO, the loss of activity was not observed. Furthermore, the loss of activity was not reversible by washing the cells. Lastly, nitrite appears to specifically target the AMO enzyme in a manner different from that of characterized inactivators of AMO.”
Substrates = food in these science-y papers
So if there was still ammonia, everything was fine.


“The trend of ammonia oxidation activity loss was correlated with the relative proportions of nitrite produced and ammonium remaining in the incubation medium after 24 h (Fig. (Fig.1B).1B). The accumulation of nitrite in the medium was proportional to the initial concentration of ammonium up to 20 mM. In incubations containing 25 to 50 mM ammonium, nitrite accumulation ceased after reaching about 21 mM. At this point, the limiting pH for ammonia oxidation, 5.7 to 6.0, was reached. The greatest losses of ammonia oxidation activity were observed with the largest concentrations of nitrite, from 15 to 21 mM, and the smallest concentrations of ammonium, from 0 to 5 mM, remaining in the medium after the 24-h incubation.”
So when the ammonia got really low and the nitrites got really high, that's when it got bad.
What does this mean for our process?


“After the incubations with nitrite, cells were sedimented, washed, and resuspended in sodium phosphate buffer for 48 h to determine if the effect of nitrite was reversible. No activity was recovered during these incubations, suggesting an irreversible inactivation of the ammonia oxidation activity by nitrite”
If the ammonia bacteria get stunted, they don't recover.

“although the interactions between ammonia- and nitrite-oxidizing bacteria are dependent upon several factors including substrate availability, O2, and pH (14), the toxic effect of nitrite on ammonia oxidizers could be ameliorated by the close physical association between the two bacterial groups as observed in soils and bioreactors (15, 18). The close physical positioning of ammonia and nitrite oxidizers in the natural environment is useful for both energetic reasons and the prevention of substrate accumulation that could be toxic or could lead to the formation of toxic by-products, especially in environments where nitrite cannot simply diffuse away (8). Therefore, this study suggests that the challenge for ammonia-oxidizing bacteria in natural systems is not only survival with an inconstant energy source but also avoidance of toxic product formation once the ammonia has been converted to nitrite”
Basically just says: The natural biological cycle protects the cells from dying to nitrite, in natural environents.
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Old 01-02-2014, 01:16 AM   #189
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All right, read that study you posted the abstract from. The one that claims that nitrite did not only NOT inhibit the Nitrosomas, but boosted it.
Some other useful things to be learned from this study - such as the fact that starved nitrosomonas will not die, but go dormant, even after starvation for MONTHS.

Source: Nitrite as a Stimulus for Ammonia-Starved Nitrosomonas europaea

“cells of N. europaea starved for ammonia were not sensitive to nitrite, either when they were starved in the presence of nitrite or when nitrite was supplied simultaneously with fresh ammonium. In the latter case, the initial ammonia-oxidizing activity of starved cells was stimulated at least fivefold”
Nitrites... good? (for AMOs)


“Under ammonium-limited conditions, the ammonia-oxidizing activity of cells of Nitrosomonas europaea is repressed by the presence of ammonium-assimilating heterotrophic bacteria and/or plant roots. Hence, ammonia-oxidizing bacteria have to withstand longer periods of ammonium starvation. To be able to profit quickly from the reappearance of fresh ammonium after a period of starvation, the population has to maintain a high ammonia-oxidizing potential.”
AMO are used to being starved and will go dormant rather than die.



“Pure cultures of ammonia-oxidizing bacteria are able to preserve their ammonia-oxidizing activities for several weeks.”
Just reiteration - AMO just go dormant


“The maintenance of a high ammonia-oxidizing capacity has also been demonstrated in mixed cultures of ammonia- and nitrite-oxidizing species (4, 14). In mixed cultures, however, repression of ammonia oxidation occurred within the first few hours after addition of fresh ammonium to cells of N. europaea that had been incubated under ammonium-limited conditions for several weeks (4). Since the nitrite-oxidizing cells of Nitrobacter winogradskyi were not able to keep pace with the ammonia-oxidizing cells, nitrite accumulated after addition of fresh ammonium. It was hypothesized that the ammonia-oxidizing cells had been inhibited by the freshly produced nitrite. Stein and Arp (12) showed that the specific loss of ammonia-oxidizing activity in cells of N. europaea deprived of ammonium was due to toxicity of accumulated nitrite in the incubation medium.”
Right here is where this paper references the paper I posted right before this one. It points out that the previous people concluded it was nitrite toxicity.


“Usually within 1 week after inoculation with N. europaea the ammonium was converted to nitrite, the sand slurries were decanted, and fresh mineral medium was added”
Just a brief aside that mentions cycle time. Nitrites appeared in about a week.


“Sand cultures of N. europaea that had been starved for 1, 2, and 3 months in their own spent medium (i.e., in the presence of 5 mM nitrite) were decanted, and fresh mineral medium containing 5 mM ammonium was added. The ammonium-starved cells started to produce nitrite immediately. During accumulation of nitrite, no repression of ammonia-oxidizing activity was observed during the first 24 h of incubation. A decline in ammonia-oxidizing activity was occasionally observed during the second day of incubation, which was likely due to ammonium limitation in the culture”
This pretty much just said that 24 hours there was less oxidation because there was less food, which seems pretty duh.


“After 1, 2, and 3 months of ammonium starvation in the absence of nitrite, the cultures received fresh medium with 5 mM ammonium. The potential ammonia-oxidizing activities were low compared to those observed in the experiment described above”
When they were starved without any nitrite, they recovered slower? Weird. Relevant to us, I don't know.


“After ammonium starvation for 4 months in spent medium containing 5 mM nitrite, all sand cultures were supplied with fresh medium containing 5 mM ammonium plus different combinations of nitrite, nitrate, and active nitrite-oxidizing cells of N. winogradskyi strain. Sand cultures that had received 5 mM nitrite in addition to 5 mM ammonium exhibited high potential ammonia-oxidizing activities for 5 h irrespective of the presence of cells of N. winogradskyi”
AMO cells still recovering instantly after FOUR MONTHS

The assumed toxicity of nitrite for ammonia-starved cells of N. europaea was not observed in the experiments described above, either when nitrite was present in the starvation medium or when it was present in the resuscitation medium. On the contrary, nitrite stimulated the ammonia-oxidizing activity of starved cells during the first few hours after addition of fresh medium with 5 mM ammonium”
This study did not obtain the same results after the previous study, and in fact concluded that nitrite helped the AMOs.


“It is hard to imagine how the end product of the second step (nitrite) could stimulate the oxidation of ammonia. However, nitrite can be reduced to NO by the enzyme nitrite reductase. Nitrite reduction to NO has been shown to occur in N. europaea”
They aren't sure why it seemed that nitrite helped the AMOs.

The observed stimulation of ammonia oxidation by nitrite in N. europaea contradicts the results obtained by Stein and Arp (13) with cells of N. europaea. The most obvious explanation for the difference is the physiological condition of the cells in the two studies. Our observations were made with ammonia-starved cells, whereas Stein and Arp (13) used late-log-phase cells for their inhibition experiments”
Acknowledging the conflict, and suggesting it was a difference in the condition of the cells.
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Old 01-02-2014, 01:35 AM   #190
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From a textbook, 2004. Source: Strict and Facultative Anaerobes: Medical and Environmental Aspects - Google Books

"Nitrosomonas eutropha can adapt to high nitrite concentrations, as higher than 40mM are required to inhibit ammonia oxidation."
Can we grow both? I haven't seen other references to Nit. Eutropha before.


"Slowly increasing the nitrite concentration (about 3mM per day) up to 100mM is not inhibitory (Zart et al 1998)."
I wasn't clear if this meant in all Nitrosomonas spp. or just Eutropha
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