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eaglesaquarium

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A question came up in the Cycle your Tank section regarding a newly cycled aquarium, with no fish.  The OP was heading out for a few weeks (let's say for the sake of this discussion 5 weeks).  The question is, is it best to leave the tank alone for the 4 weeks, or to have an uninitiated individual (aka, extreme newbie) dose the tank with ammonia, as per whatever instructions might be left?
 
 
 
The question might be better asked this way:  What are the pros and cons of dosing ammonia versus the pros and cons of not dosing ammonia?
 
 
As always in the scientific section, please try to provide scientific evidence for your viewpoint.
 

Byron

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I posted some comments in the other thread before I realized you had started this one, so I will just repeat the data.  As noted there, this excerpt comes from some research I carried out a couple of years back to prepare an article on bacteria in freshwater aquaria for another forum.  References [square bracket numbers in the text] are added at the end.
 
Before copying the excerpt, my suggestion is to not add ammonia once the tank has "cycled."  The nitrifying bacteria does not die off but in the absence of "food" enters into a state of hibernation in a sense.  Obviously the length of time affects how quickly the bacteria rebound, so it is best not to overload the system afterwards.  But the risks of inadvertent addition of ammonia is worth considering.
 
Nitrifying Bacteria
 
Nitrification is the oxidation of ammonia/ammonium to nitrite and then the subsequent oxidation of nitrite to nitrate; this is performed by two groups of bacteria known collectively as nitrifying bacteria or nitrifiers.  True nitrifying bacteria are autotrophs; they use chemosynthesis to manufacture their energy by using oxygen plus nitrogenous waste (ammonia or nitrite) and carbon (from CO2).  There are several different bacterium species involved, all in the family Nitrobacteraceae, that carry out this function in soil, and it used to be thought that these, particularly Nitrosomonas europa and Nitrobacter, were the nitrification bacteria in freshwater.  But Dr. Timothy Hovanec led the team of scientists that proved this to be a mistaken assumption.  Ammonia is converted to nitrite by bacteria of the Nitrosonomas marina-like strain [2] and nitrite is converted to nitrate by bacteria closely related to Nitrospira moscoviensis and Nitrospira marina. [3]  With several subsequent scientific studies by other scientists on wastewater nitrifying bacteria this data is now accepted and confirmed scientific fact.
 
Once established, the population of these bacteria in an aquarium will be in direct proportion to the amount of ammonia or nitrite respectively.  Nitrifying bacteria require 12-32 hours to multiply, which they do by binary division [each bacterium divides into two bacteria].  Nitrosomonas multiply in less time (12+ hours) while Nitrospira require more time (up to 32 hours).  In a new aquarium, it can take up to eight weeks for the bacteria populations to reach a level capable of eliminating ammonia and nitrite.
 
Scientific studies have also now proven that Nitrospira are inhibited and cannot multiply in water that contains significant concentrations of ammonia, and evidence exists to suggest that existing populations of Nitrospira actually become dormant when ammonia is present in high concentrations.  Kim et al. (2006) determined that with an active ammonia [NH3] level of 0.7 mg/l (=ppm) Nitrospira bacteria experienced a decrease of 50% effectiveness, resulting in an accumulation of nitrite. [4]
 
The pH has a direct effect on nitrifying bacteria.  These bacteria operate at close to 100% effectiveness at a pH of 8.3, and this level of efficiency decreases as the pH lowers.  At pH 7.0 efficiency is only 50%, at 6.5 only 30%, and at 6.0 only 10%.  Below 6.0 the bacteria enter a state of dormancy and cease functioning. [5]  Fortunately, in acidic water (pH below 7.0) ammonia automatically ionizes into ammonium which is basically harmless.  And since nitrite will not be produced when the ammonia-oxidizing bacteria are in “hibernation,” this decrease in their effectiveness poses no immediate danger to the fish and other life forms.
 
Temperature also affects the rate of growth of nitrifying bacteria.  It will be optimal at a temperature between 25 and 30C/77 and 86F.  At a temperature of 18C/64F it will be 50%.  Above 35C/95F the bacteria has extreme difficulty.  At both 0C/32F (freezing) and 100C/212F (boiling) the bacteria die.
 
These bacteria cannot survive drying out; without water, they die.  Tap water with chlorine or chloramine will kill these bacteria.  Antibacterial medications will negatively impact the nitrifying bacteria to varying degrees.
 
[2] Paul C. Burrell, Carol M. Phalen, and Timothy A. Hovanec, “Identification of Bacteria Responsible for Ammonia Oxidation in Freshwater Aquaria,” Applied and Environmental Microbiology, December 2001, pp. 5791-5800.
 
[3] Hovanec, T. A., L. T. Taylor, A. Blakis and E. F. DeLong, “Nitrospira- Like Bacteria Associated with Nitrite Oxidation in Freshwater Aquaria,” Applied and Environmental Microbiology, Vol. 64, No. 1, pp.  258-264.
 
[4] Kim, D.J., D.I. Lee and J. Keller (2006), “Effect of temperature and free ammonia on nitrification and nitrite accumulation in landfill leachate and analysis of its nitrifying bacterial community by FISH,” Bioresource Technology 97(3), pp. 459-468.
 
[5] Kmuda, “Aquarium Bacteria and Filtration Manifesto,” Parts 1 and 2, OscarFish website.
 

Chad

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My first issue is isolating the species of bacteria in aquaria as being consistent. If they aren't then we're left making assumptions that may or may not apply. Certainly we may say or think we're making a safe bet by guessing they are, but in actuality if they aren't one species may be quite different than another in terms of dormancy and alternative food sources.
 

TwoTankAmin

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Tcamos, since this is the scientific section, would it be too much bother to ask you to provide some science to support what you are saying?
 
As for what bacteria may be at work, lets make it simple here. Lets not look at anything but those which process ammonia. We can leave out all the others for now. We also do not have to spend much time on the nitrite oxidizers because they are way less diverse. And since we are talking about how long they can last under times of low or no nutrients, let us tryto focus on that. Fortunately this is a pet subject of mine over the years and I have a lot od research bookmarked, so I will offer one side of this issue and then allow you to provide the science which contradicts or shows what I have offered to have been supplanted by subsequent work.
 
Lets start here:
 
Strategies of aerobic ammonia-oxidizing bacteria for coping with nutrient and oxygen fluctuations
http://onlinelibrary.wiley.com/doi/10.1111/j.1574-6941.2006.00170.x/full
 
This one is a lovely piece of work which basically reviews a lot of the literature on this topic. So it presents information from multiple studies and researchers which caver an assortment of AOB and strains thereof. As you read through this paper you will see there is one common thread. All of the various autotrophic ammonia oxidizers have a strategy that lets them cope with a withdrawal of ammonia as well as other things such as oxygen or carbon. So what this paper does is give you conclusions based on their revue of many many studies on the subject. I did not count but I think this paper will give you about 40 other studies on the topic. And here is the first paragraph of the conclusion:
 
From this review it is clear that AOB possess several physiological traits that can be advantageous for their survival under conditions of variable substrate and oxygen supply. Moreover, AOB possess a number of enzymological and molecular mechanisms that allow them to maintain the state of their cells under starvation such that ammonia oxidation can start within minutes and at high rates after substrate or oxygen depletion. Furthermore, within the AOB groups, differences exist in adaptation to and competitiveness under conditions of high or low ammonia or oxygen concentrations. In addition, they seem to be able to communicate through cell-to-cell signalling and to move towards a more favourable environment.
 
So I guess I have started out with about 40 odd research papers which do not support your opinion. But lets move on from here and look at this
 
Growth at Low Ammonium Concentrations and Starvation Response as Potential Factors Involved in Niche Differentiation among Ammonia-Oxidizing Bacteria
http://aem.asm.org/c...68/10/4751.full
 
This study is similar to the first one as it reviews a lot of the literature on the subject. Its focus is on how two different strains of AOB which have differing substrate affinity for ammonia react to adverse conditions and how they fare vs plants in competing at lower ammonia levels. These are AOB from the same division as those found in aquaria. Basically, it concludes that one strain out competes another during starvation and subsequent recover, the fact remains that in the end there is recovery. However the important take away from all this is that in an aquarium, the AOB that dominate are the ones with a lower substrate affinity as the amount of ammonia generated by a tank is low. And this study clearly shows that there are low affinity strains of AOB that will survive low and no ammonia levels for some time and then recover rapidly.
 
 
Moving on again, lets take a look at this research:
 
Cell density-regulated recovery of starved biofilm populations of ammonia-oxidizing bacteria.
http://Cell density-regulated recovery of starved biofilm populations of ammonia-oxidizing bacteria
 
This study is an older one, from 1997, but it is still to the point. They looked at AOB in a biofilm in two ways- in aqautic and in soil environments. They starved them of ammonia for 42 days and then reintroduced it and measure how long it took for nitrite to begin rising. As you will see, in either case the bacteria were able to recover rapidly. The Study focussed on Nitrosomonas europea. They attribute the ability to recovery more rapidly than expected to the biofilm which host the nitrifiers and many other bacteria. So once again we see rapid recovery within the nitrosomonas cluster.
 
And lets look at how broad the ability of various AOB strains etc. is in terms of recovery from ammonia starvation. Here is a study on the subject for salt water strains of AOB. This one goes all the back to 1983 indicating that this phenomenon has been researched for many decades:
 
Nitrification in closed seawater culture systems: Effects of nutrient deprivation
http://www.sciencedi...044848683902934
 
 
Abstract
Nitrifying bacteria in closed, conditioned seawater culture systems remained active for periods up to 16 weeks after nitrogen input to the systems had been terminated by removing all the stocked fish. The amounts of ammonia that were oxidized in 24 h decreased by 8, 47, 54, 62, and 91% after 1, 2, 4, 8, and 16 weeks, respectively, of nutrient deprivation, as compared with values measured before the onset of deprivation. As expected, decreases in nitrite oxidation were nearly identical with those for ammonia.
Pre-deprivation nitrification rates were recovered rapidly, upon the reintroduction of ammonia into the water; at least 70% of the initial ammonia-oxidizing capacities were regained within 48 h, even in systems that had been deprived of nutrients for 8 weeks.
 
 
 
A lot of the key to understanding AOB and ammonia starvation and recovery is to see it applies, albeit with differing starvation and recivery times, to a very wide variety of AOB, not just those which may be in aquariums. This is an ability shared by all of the autotrophic AOB. Here are several more studies which support this:
 
Influence of Starvation on Potential Ammonia-Oxidizing Activity and amoA mRNA Levels of Nitrosospira briensis
http://aem.asm.org/c.../71/3/1276.full
 
 
In nature, AOB often face longer periods of NH4+ starvation and limitation due to low nitrogen input, low mineralization rates, or competition with other AOB (8), heterotrophic bacteria (48, 49), or plants (5, 6, 50). In order to respond rapidly when NH4+ becomes available, AOB must maintain their ability to oxidize NH4+ during these periods.
With the exception of a few marine strains within the genus Nitrosococcus (of the γ-subclass of the Proteobacteria), all known AOB belong to a distinct clade within the β-subclass of the Proteobacteria (13), which comprises 11 clusters (37). By using 16S rRNA gene and more recently amoA gene sequencing, directly from environmental samples, the distribution of the members of the different clusters of AOB has been correlated to the characteristics of the environments (29, 37). The starvation behavior of several AOB belonging to different phylogenetic groups has previously been investigated. Nitrosomonas europaea affiliated with Nitrosomonas cluster 7—a group of AOB detected in environments with high NH4+ availability like wastewater (36, 40, 51)—rapidly became active again after periods of starvation in batch and retentostat experiments (8, 31, 46, 52), and the marine AOB, Nitrosomonas cryotolerans, showed a similarly rapid response to the presence of ammonia (22, 23, 24). On the other hand, members of Nitrosomonas cluster 6a (Nitrosomonas oligotropha group), often found in freshwater environments (7, 12, 43), and Nitrosospira briensis, often found in terrestrial habitats, regain their activity slower than Nitrosomonas europaea after long-term starvation of 10 weeks or 4 months (8, 32).
 
And here is another which is only an abstract:
 
Long-term storage and subsequent reactivation of aerobic granules
http://www.sciencedi...960852408002009
 
 
Abstract


This study investigated a seven month storage and the subsequent reactivation of aerobic granules. The granule size and structure integrity were remained during storage, whereas some cavities and pleats appeared on the surface and further deteriorated the settleability. Along with the reactivation, the physical characteristics and microbial activities of aerobic granules were gradually improved. Activities of heterotrophs and nitrifiers can be fully recovered within 16 days and 11 days, respectively. Nitrifiers decayed slower during storage and reinstated rapider during reactivation than heterotrophs. In fresh aerobic granules, the dominated ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) were Nitrosomonas and Nitrospira, respectively. During storage, the initially dominated populations decayed rapider than the initially less dominated ones. Extracellular polymeric substances (EPS) significantly decreased within the first month, and then gradually accumulated during the last six months storage. Accumulation of EPS was an effective strategy for maintaining structural integrity of aerobic granules during long-term storage.
Again we see AOB being starved of ammonia for 7 months, not the one month or so metioned in the forum thread that fostered this discussion. And it looked at both Nitrosomonas and Nitrosospira.
 
And one of my favorite pieces of research on the subject of bacterial survival is this one which deals with soil based AOB:
 
Survival of bacterial DNA and culturable bacteria in archived soils from the Rothamsted Broadbalk experiment
http://www.sciencedi...038071707004683
 
 
In a preliminary study, to establish if dried soils can provide a historical record of bacterial communities, samples from the Broadbalk soil archive dating back to 1868 were investigated and plots treated with either farmyard manure (FYM) or inorganic fertilizer (NPK) were compared. As anticipated, the processes of air-drying and milling greatly reduced bacterial viability whilst DNA yields declined less and may be preserved by desiccation. A higher proportion of culturable bacteria survived the archiving process in the FYM soil, possibly protected by the increased soil organic matter. The majority of surviving bacteria were firmicutes, whether collected in 2003 or in 1914, but a wide range of genera was detected in DNA extracted from the samples using PCR and DGGE of 16S rRNA genes. Analysis of DGGE band profiles indicated that the two plots maintained divergent populations. Sequence analysis of bands excised from DGGE gels, from a sample collected in 1914, revealed DNA from α- and β-proteobacteria as well as firmicutes.
 
Basically what the above is saying is they found some amount of culturable AOB from the α- and β-proteobacteria which are the ones which contain the autotrophic ammonia oxidizers. They are indicating some culturable bacteria was present in sample close to 100 years old. It may have been a small amount, but it was there.  The bacteria can survive in dried dirt basically. And this leads me to conclude with the following from Dr. Timothy Hovanec:
 
 
Nitrifying bacteria can live in a bottle for a while. Many think that nitrifying bacteria cannot live in a bottle and will say the reason is because nitrifying bacteria don’t form spores like other bacteria. This is a half-truth. Nitrifying bacteria don’t form spores, but that doesn’t mean they can’t last in a bottle (think about it – if nitrifying bacteria could not survive poor conditions, how would they have survived for millions of years?) They can live in a bottle but under optimal conditions, and the time period is about one year. The nitrifying bacteria don’t die in the bottle; their activity level drops and eventually it becomes so low that there is little measurable positive effect when they are poured into the aquarium water. Provided the nitrifying bacteria in the bottle were not subject to bad environmental conditions (see the next paragraph), they can last about one year in a bottle.
from http://www.drtimsaquatics.com/nitrifying-bacteria-mixtures-work-provided
 
There is one thing on which we do have some agreement here is the fact that once inside a closed environment such as an established aquarium that a given identified strain of AOB may adapt to that tank. By this I mean the strain will be very similar, but not 100% identical to those recorded in bacterial banks and identified taxonomically. But this is not uncommon in the literature. What we see are terms like Nitrosomas europea-like indicating a strain is very similar but not identical. However, this doesn't seem to change how they function overall only how they match up genetically.
 
I believe that the exact microbial "roster" in any given aquarium is unique to it. However, I do not believe that this fact mean the bacteria present from tank to tank do not share similar functions and capabilities. And then there is also limit to the variety of known autotrophic AOB. It isn't as if there are 100s of identified autrophic ammonia oxidizing bacteria. But what is also clear is that in any given tank there is likely more than one type of AOB at work in the biofilm.
 
Microenvironments and distribution of nitrifying bacteria in a membrane-bound biofilm
http://ftp.mpi-bremen.de/pub/molecol_p/armin/Publ_Nitrifikation/Schramm_EM_2000.pdf
 
 
Summary
The distribution of nitrifying bacteria of the genera Nitrosomonas, Nitrosospira, Nitrobacter and Nitrospira was investigated in a membrane-bound biofilm system with opposed supply of oxygen and ammonium.
Gradients of oxygen, pH, nitrite and nitrate were determined by means of microsensors while the nitrifying populations along these gradients were identified and quantified using fluorescence in situ hybridization (FISH) in combination with confocal laser scanning microscopy. The oxic part of the biofilm which was subjected to high ammonium and nitrite concentrations was dominated by Nitrosomonas
europaea-like ammonia oxidizers and by members of the genus Nitrobacter. Cell numbers of Nitrosospira sp. were 1±2 orders of magnitude lower than those of N. europaea. Nitrospira sp. were virtually
absent in this part of the biofilm, whereas they were most abundant at the oxic±anoxic interface. In the totally anoxic part of the biofilm, cell numbers of all nitrifiers were relatively low. These observations
support the hypothesis that N. europaea and Nitrobacter sp. can out-compete Nitrosospira and Nitrospira spp. at high substrate and oxygen concentrations. Additionally, they suggest microaerophilic
behaviour of yet uncultured Nitrospira sp. as a factor of its environmental competitiveness.
 
Finally,let us not forget that there is strong evidence that ammonia oxidizing archaea are now known to play a role in ammonia oxidation. This is clear for sw aquatic environments and for soil as well. It is less clear in terms of fw where how much work might be done by any AOA vs AOB is still under debate. What does appear certain is there are some AOA present in fw tanks even if they are not the primary ammonia oxidizers. These AOA are a bit less complex than the the AOB, but they are also autotrophic which means they need survival strategies since they do not form spores. They reproduce in a similar fashion to the AOB.
 
So tcamos, I will wait patiently for you to provide the type of science that this forum requires to support your point that there might be some autotrophic nitrifiers at work in tanks that, unlike virtually all of the others, is unable to survive for any meaningful amount of time in the absence of ammonia. That cannot even last for a month as in the example that engendered this discussion.
 
I am now suffering from deja vu. Several years ago in the very same forum this very same discussion took place. It was more centered around the bottled bacteria products and if they could work and if the bacteria could even survive in a bottle. I quoted some of the same studies back then as I have included in this post. The hallmark of those discussions were simple. I quoted study after study and those who wanted to argue against them quoted virtually none. If one looks at the research I have linked to above and then looks at the references listed what you will find are 100s of studies showing the bacteria can survive ammonia starvation and can recover pretty rapidly in most cases.
 
tcamos- the ball is now in your court. All I would ask is you provide links to the research to support what you have said as I have done with what i have contended. Show us what autotrophic oxidizing bacteria cannot survive ammonia starvation for a month let alone much longer and then it would help to show that these or a close relative is likely to be at work in established aquaia,
 

TwoTankAmin

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There is a lot more information the research has shown in respect to autotrophic AOB and their ability to survive ammonia startvation for extended periods and how they recover. Lets take a quick look at another study from The American Society for Microbiology's Journal of Bacteriology published in 2012
 
The Divergent AmoC3 Subunit of Ammonia Monooxygenase Functions as Part of a Stress Response System in Nitrosomonas europaea
 
 
Previous work suggested a possible functional role for amoC3 as part of the σE stress response regulon during the recovery of N. europaea from extended ammonia starvation, thus indicating its importance during the exit of cells from starvation. We here used global transcription analysis to show that expression of amoC3 is part of a general poststarvation cellular response system in N. europaea. We also found that amoC3 is required for an efficient response to some stress conditions, as deleting this gene impaired growth at elevated temperatures and recovery following starvation under high oxygen tensions....
 
Starvation and nutrient limitation are stresses common to all bacteria that compete for resources in the environment. For example, ammonia-oxidizing bacteria must compete with plants and other microorganisms that rely on the assimilation of ammonia for biosynthesis (48, 49). To survive periods of ammonia starvation and succeed in direct competition with other organisms, ammonia-oxidizing bacteria have developed several physiological traits such as high viability and low maintenance energy requirements during starvation (22, 46), stable macromolecular components (6, 52), and cellular mechanisms that enable cells starved of ammonia for extended periods of time to initiate ammonia oxidation activity within minutes of reexposure to ammonia (7, 12, 23, 52).
from http://jb.asm.org/content/194/13/3448.full
 
This study investigated how the AOB recover from periods of ammonia starvation and from heat stress. From the above paragraphs in the Abstract and the  Introduction we can see that the ability of the autorophic AOB to survive and then recover from ammonia starvation is considered an intrinsic ability of these bacteria and what contributes to this mechanism.
 
As one starts to read trough the literature on this subject this ability is discussed and explained over and over. I do not claim to have read everything out there on the subject, However, I have looked at a fair number of both abstracts and full studies and the one thing I have not come across is a single study or paper which states that any one of the individual AOB types was unable to cope with starvation or unable to recover from it within a reasonable time period. By reasonable I am talking about months not years in terms of starvation and being able to effect a reasonably rapid recovery. The studies on some of the soil AOB have shown there can be enough bacteria that is viable after years of storage in soil banks to  be culturable even after decades, this does not mean that it recovers to being a fully functional colony but that there is sufficient viable bacteria to act as seeding for a new colony.
 
However, when it comes to aquaria and bacterial survival, we are not talking about years. Here we are normally dealing with things in terms of a number of weeks or perhaps a few months. For us in the hobby these are really the relevant periods. After all, how many people keep a tank devoid of an ammonia source up and running for 3 months let alone a year? In addition, in aquaria, we are working with AOB which are best adapted to low ammonia concentrations.
 
When one reviews the literature they will also discover that the biofilm in which the nitrifiers live also contributes to the ability of bacteria to resist adverse conditions, including ammonia deprivation. In fact, it is virtually impossible to come to any real understanding of the microbiology in tanks without having an understanding of the biofilm and its role in everything. But this is a topic for discussion in a different thread.
 

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