HighlandReefer
Team RC
You're welcome. 
Interesting article regarding current knowlege of anammox bacteria & the biochemistry
- Thread starter HighlandReefer
- Start date
:thumbsup:
Great finds, Cliff.
Great finds, Cliff.
HighlandReefer
Team RC
Thanks Randy.
It's always nice to get a compliment from my mentor.
It's always nice to get a compliment from my mentor.
After reading the whole study once I,m not sure it's very relevant to aquarium applications. Specialized industrial reactors involving nitrite injections and many other manipulations are used in waste water studies and aplications noted and measured for cost efficiency vs methanol reactors.
Naturally occuring amanonox activity happens predominantly at the edge of sulfur reducing anoxic regions which is where the ammonium for the process originates, as I read it. So would you need SO4 reduction and concomitant sulfide and hydrogen sulfide production for anamonox bacteria to play a significant role in a closed system with or without a deep sand bed or plenum? It was first discovered in the large anoxic basin known as the Black Sea and subsequently in some large upwelling systems in the ocean where it is hypothesized that ammonium wells up from the sulfur reducing regions where it is produced along the way to mineralization. The tank water would normally be low in ammonium in a healthy aquarium. since oxidation via the usual aerobic nitrifying process would likely occur first, so it seems a source of ammonium to very hypoxic areas would be needed to encourage anamonox activity . I don't think that is worth the effort or the risks at least at the stage of knowledge now available . The anamonox bacteria involved are very slow growers(11 to 12 days for division) as far as bacteria go so any efect on nitrogen reduction would likely be very slow.
It's difficult to cross over waste water applications to biotypes like aquariums but the studies can be thought provoking .Personally, I don't use them as baseline material for aquariums.
Now for winners and losers, vodka and vinegar vs vinegar only. IMO, it's a tie.
Ethanol oxidizes to acetic acid(vinegar without the dilution) ,so it sources acetate after an extra step or two as well . These steps don't result in discernible harm and may or may not be of benefit. I'd guess they cause no significant benefit or harm based on my 3 year experience.
I'll have to read the study again because I didn't find a clear indication that ethanol suppresses anamonox bacteria but even if it does I'm not sure it matters in an aquarium since there may not be any significant number of them to start with.
Ethanol is also a neutral ph additive as opposed to vinegar with a low ph and CO2 additions.
I prefer bolus dosing as opposed to incremental dosing via dosing pump and timers needed with larger amounts of vinegar to mitigate precipitous ph effects. Bouls dosing is the routine I've followed for 3 years with satisfying results, a complete change over would change bacterial populations .
While I'm always looking for opportunities for improvement as per the shift to 25% vinegar from all vodka several years ago, a total change of carbon source and resuting changes in the bacterial cultures in my aquarium are not a hand I want to play right now..
I also dose some vodka at night without worry about introducing more CO2.
Bolus dosing may encourage more anaerobic denitrifiction activity than incremental dosing according to one study.
I haven't seen anything that would ease my reluctance to switch to only vinegar in my system in the amnamonox writings but I think vinegar only makes good sense for some systems and would be inclined to try it if I was just starting if for no other reason than to skip the extra bacterial steps ethanol needs to go to acetate andto see how the animals in t4h tank reacted vs the curent vodka/ vinegar method.
Vodka does cost more but not much when you don't count the extra water.
The vodka I use is $14 for 1.75 liters(about 59 ounces) ,40% of that is ethanol roughly 23.6 ounces ,for a per ounce cost of 59 cents.
I use the store brand vinegar in the economy size,It;s the cheapest around here @ $4 for 154 ounces, 95% of which is water leaving 7.7 ounces of acetic acid for $4 or 52 cents per ounce.
The biopellets vs vinegar or vodka and vinegar favors the ethanol and acetic acid or acetic acid ,imo.
Either ofwhich can be dosed via an automated system. . Biopellets are polymers( carbohydrates) . They are more complex than the ethanol or vinegar. Polymers go through more bacterial steps and create more by products on the route to acetate . They go to monomers, sugars. I prefer to avoid polymers and monomers because of bad reactions by some corals to even very small doses of sugar.
Either ofwhich can be dosed via an automated system. . Biopellets are polymers( carbohydrates) . They are more complex than the ethanol or vinegar. Polymers go through more bacterial steps and create more by products on the route to acetate . They go to monomers, sugars. I prefer to avoid polymers and monomers because of bad reactions by some corals to even very small doses of sugar.
I personally, strongly prefer vodka and vinegar or only vinegar to biopellets. Either of these carbon sources can be dosed via dosing pump or with kalk top off if incremental vs bolus dosing is preferred.
The amount or organic carbon going into your tank is in your direct control With the pellets it depends on flow, guess workd on the amount of pelets to use, varying types of pellets,varying reactors, etc.
The biopellets are polymers( carbohydrates) . They are more complex and go through more cascading bacterial activity with more types of bacteria and by products involved along the route to acetate generation. The polymers go to monomers( sugars) which have been harmful to some corals based on my experience and that of others when dosing even small amounts of sugar.
The amount or organic carbon going into your tank is in your direct control With the pellets it depends on flow, guess workd on the amount of pelets to use, varying types of pellets,varying reactors, etc.
The biopellets are polymers( carbohydrates) . They are more complex and go through more cascading bacterial activity with more types of bacteria and by products involved along the route to acetate generation. The polymers go to monomers( sugars) which have been harmful to some corals based on my experience and that of others when dosing even small amounts of sugar.
HighlandReefer
Team RC
Anammox bacteria: from discovery to application
J. Gijs Kuenen1 About the author
http://www.nature.com/nrmicro/journal/v6/n4/abs/nrmicro1857.html
Abstract
Anaerobic ammonium oxidation (anammox) bacteria, which were discovered in waste-water sludge in the early 1990s, have the unique metabolic ability to combine ammonium and nitrite or nitrate to form nitrogen gas. This discovery led to the realization that a substantial part of the enormous nitrogen losses that are observed in the marine environment — up to 50% of the total nitrogen turnover — were due to the activity of these bacteria. In this Timeline, Gijs Kuenen recalls the discovery of these unique microorganisms and describes the continuing elucidation of their roles in environmental and industrial microbiology.
--------------------------------------------------------------------------------
18.04.2005
Advanced measurements off the coast of Namibia give a new explanation for the extremely efficient nitrogen removal from the oxygen poor areas of the ocean.
http://www.mpi-bremen.de/en/Anammox_Bacteria_produce_Nitrogen_Gas_in_Oceans_Snackbar.html
From it:
"The researchers discovered this type of bacteria for the first time a few years ago in the oxygen poor Black Sea and now also in the open ocean. This discovery has major consequences for our understanding of the global nitrogen cycle. The Benguela current system leads to upwelling of nutrient-rich cold water off the coast of Namibia and acts as a kind of snackbar in the tropical ocean, which is visited by many animals including giant whales. The newly discovered anammox bacteria remove ammonium from the ocean, which as a result can not be taken up anymore by other organisms. Algae and cyanobacteria only partly succeed in fixing the released nitrogen gas to form new nutrients that can be fed into the nutrient cycle again."
"Denitrification.
The measurements refute the dominating theory that oceanic nitrogen loss results from the activity of bacteria that convert nitrate (via nitrite) with organic matter to nitrogen gas in the absence of oxygen (denitrification). In fact, the researchers recently discovered that anammox bacteria can use organic matter to convert nitrate into nitrite (much better than denitrifying bacteria). This new finding even increases the importance of anammox bacteria in the ocean.
New species
The anammox bacteria discovered in the Atlantic Ocean are closely related to the bacterial species Scalindua sorokinii, which was recently discovered in the Black Sea (Kuypers et al., 2003 Nature 8 April). Like their relatives in the Black Sea, the Namibian anammox bacteria contain unique ladder molecules (Damsté et al., Nature 17 October 2002) in the membrane surrounding a special prokaryotic organel in which ammonium is converted to nitrogen gas. These ladder molecules are ether bound in the membrane. This property was believed to be restricted to the Archaea, the ‘ancient’ bacteria."
-------------------------------------------------------------------------------------
Making a Progress to Speed up the Nitrification and Denitrification
Processes in Novel Biosorption Activated Media: Can Archaea be in
Concert with Anammox?
www.omicsonline.org/2155-9821/2155-9821-1-103e.pdf
From it:
"There are various players in the nitrogen cycle and the diversity and
functions of the microorganisms involved in nitrification and denitrification
is quite complex [7]. The general metabolic path of historical assumptions
is pretty limited from ammonia to nitrite, to nitrate, and to nitrogen
Gas (N2), which has been widely acknowledged. In the presence of
ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB),
ammonium is converted to nitrite and further to nitrate. These two reactions
are collectively called nitrification. Denitrification, conversely, performed
by denitrifying community, is an anaerobic respiration process
using nitrate as a final electron acceptor and result in stepwise microbiological
reduction of nitrate, nitrite, nitric oxide (NO), nitrous oxide (N2O)
and nitrogen gas. On the top of this understanding, it is becoming clear
that denitrifying fungi, nitrifying archaea, anammox bacteria, aerobic denitrifying
bacteria and heterotrophic nitrifying microorganisms are all key
players together in the nitrogen cycle [7]."
"Recently, there are two significant discoveries for nitrogen cycle: 1) the
occurrence of ammonia oxidizing archaea (AOA). In some environments,
AOA dominate the ammonia-oxidizing community in the nitrification
process. 2) The occurrence of anaerobic ammonia oxidation (anammox)
reaction. In this biological process, nitrite and ammonium can be converted
directly into dinitrogen gas: NH4
+ + NO2
- → N2 + 2 H2O. Such a shortcut
of nitrogen cycle has been proven to exist and can be portrayed holistically
in Figure 1 in which the biogeochemical nitrogen network associated with
possible microbial species and reaction pathways may be connected [9]."
"In laboratory-scale analysis, enrichment of anammox bacteria from marine
environment can be carried out using a continuous culture system
[35]. Tsushima, et al. (2007) [36] adopted a rotating disk reactor (RDR)
biofilm in semi-batch cultures as a means to carry out such an enrichment
of cultures and to quantify anammox by using real-time PCR. Phylogenetic
analysis revealed that all the detected clones were related to the previously
reported anammox bacteria, Candidatus Brocadia anammoxidans
(AF375994), with 92% sequence similarity [36]."
"Besides, in aquaria
and recirculating aquaculture systems, the accumulation of ammonia, an
end product of protein metabolism in aquatic life, must be removed because
of its toxicity to fish community. Urakawa et al. (2008) [18] started
investigating the phylogenetic diversity of AOA and AOB in aquarium
biofiltration systems to explore the nitrification of an engineered filtration
media system. The findings imply that the phylogenetic diversity and
species richness of AOA are greater than those of AOB and temperature
is a key factor influencing the population structure and diversity of AOA
and AOB in aquarium biofiltration systems. Even AOB can degrade halogenated
compounds in the reclamation of wastewater [38]. Natural and
artificial wetlands may be used as an ecological engineering unit to treat
stormwater and wastewater. Anammox and anaerobic methane oxidation
(ANME coupled to denitrification) with nitrite as electron acceptor are
two of the most recent discoveries in the microbial nitrogen cycle of wetland
ecosystem [29]."
------------------------------------------------------------------------------------
J. Gijs Kuenen1 About the author
http://www.nature.com/nrmicro/journal/v6/n4/abs/nrmicro1857.html
Abstract
Anaerobic ammonium oxidation (anammox) bacteria, which were discovered in waste-water sludge in the early 1990s, have the unique metabolic ability to combine ammonium and nitrite or nitrate to form nitrogen gas. This discovery led to the realization that a substantial part of the enormous nitrogen losses that are observed in the marine environment — up to 50% of the total nitrogen turnover — were due to the activity of these bacteria. In this Timeline, Gijs Kuenen recalls the discovery of these unique microorganisms and describes the continuing elucidation of their roles in environmental and industrial microbiology.
--------------------------------------------------------------------------------
18.04.2005
Advanced measurements off the coast of Namibia give a new explanation for the extremely efficient nitrogen removal from the oxygen poor areas of the ocean.
http://www.mpi-bremen.de/en/Anammox_Bacteria_produce_Nitrogen_Gas_in_Oceans_Snackbar.html
From it:
"The researchers discovered this type of bacteria for the first time a few years ago in the oxygen poor Black Sea and now also in the open ocean. This discovery has major consequences for our understanding of the global nitrogen cycle. The Benguela current system leads to upwelling of nutrient-rich cold water off the coast of Namibia and acts as a kind of snackbar in the tropical ocean, which is visited by many animals including giant whales. The newly discovered anammox bacteria remove ammonium from the ocean, which as a result can not be taken up anymore by other organisms. Algae and cyanobacteria only partly succeed in fixing the released nitrogen gas to form new nutrients that can be fed into the nutrient cycle again."
"Denitrification.
The measurements refute the dominating theory that oceanic nitrogen loss results from the activity of bacteria that convert nitrate (via nitrite) with organic matter to nitrogen gas in the absence of oxygen (denitrification). In fact, the researchers recently discovered that anammox bacteria can use organic matter to convert nitrate into nitrite (much better than denitrifying bacteria). This new finding even increases the importance of anammox bacteria in the ocean.
New species
The anammox bacteria discovered in the Atlantic Ocean are closely related to the bacterial species Scalindua sorokinii, which was recently discovered in the Black Sea (Kuypers et al., 2003 Nature 8 April). Like their relatives in the Black Sea, the Namibian anammox bacteria contain unique ladder molecules (Damsté et al., Nature 17 October 2002) in the membrane surrounding a special prokaryotic organel in which ammonium is converted to nitrogen gas. These ladder molecules are ether bound in the membrane. This property was believed to be restricted to the Archaea, the ‘ancient’ bacteria."
-------------------------------------------------------------------------------------
Making a Progress to Speed up the Nitrification and Denitrification
Processes in Novel Biosorption Activated Media: Can Archaea be in
Concert with Anammox?
www.omicsonline.org/2155-9821/2155-9821-1-103e.pdf
From it:
"There are various players in the nitrogen cycle and the diversity and
functions of the microorganisms involved in nitrification and denitrification
is quite complex [7]. The general metabolic path of historical assumptions
is pretty limited from ammonia to nitrite, to nitrate, and to nitrogen
Gas (N2), which has been widely acknowledged. In the presence of
ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB),
ammonium is converted to nitrite and further to nitrate. These two reactions
are collectively called nitrification. Denitrification, conversely, performed
by denitrifying community, is an anaerobic respiration process
using nitrate as a final electron acceptor and result in stepwise microbiological
reduction of nitrate, nitrite, nitric oxide (NO), nitrous oxide (N2O)
and nitrogen gas. On the top of this understanding, it is becoming clear
that denitrifying fungi, nitrifying archaea, anammox bacteria, aerobic denitrifying
bacteria and heterotrophic nitrifying microorganisms are all key
players together in the nitrogen cycle [7]."
"Recently, there are two significant discoveries for nitrogen cycle: 1) the
occurrence of ammonia oxidizing archaea (AOA). In some environments,
AOA dominate the ammonia-oxidizing community in the nitrification
process. 2) The occurrence of anaerobic ammonia oxidation (anammox)
reaction. In this biological process, nitrite and ammonium can be converted
directly into dinitrogen gas: NH4
+ + NO2
- → N2 + 2 H2O. Such a shortcut
of nitrogen cycle has been proven to exist and can be portrayed holistically
in Figure 1 in which the biogeochemical nitrogen network associated with
possible microbial species and reaction pathways may be connected [9]."
"In laboratory-scale analysis, enrichment of anammox bacteria from marine
environment can be carried out using a continuous culture system
[35]. Tsushima, et al. (2007) [36] adopted a rotating disk reactor (RDR)
biofilm in semi-batch cultures as a means to carry out such an enrichment
of cultures and to quantify anammox by using real-time PCR. Phylogenetic
analysis revealed that all the detected clones were related to the previously
reported anammox bacteria, Candidatus Brocadia anammoxidans
(AF375994), with 92% sequence similarity [36]."
"Besides, in aquaria
and recirculating aquaculture systems, the accumulation of ammonia, an
end product of protein metabolism in aquatic life, must be removed because
of its toxicity to fish community. Urakawa et al. (2008) [18] started
investigating the phylogenetic diversity of AOA and AOB in aquarium
biofiltration systems to explore the nitrification of an engineered filtration
media system. The findings imply that the phylogenetic diversity and
species richness of AOA are greater than those of AOB and temperature
is a key factor influencing the population structure and diversity of AOA
and AOB in aquarium biofiltration systems. Even AOB can degrade halogenated
compounds in the reclamation of wastewater [38]. Natural and
artificial wetlands may be used as an ecological engineering unit to treat
stormwater and wastewater. Anammox and anaerobic methane oxidation
(ANME coupled to denitrification) with nitrite as electron acceptor are
two of the most recent discoveries in the microbial nitrogen cycle of wetland
ecosystem [29]."
------------------------------------------------------------------------------------
Last edited:
HighlandReefer
Team RC
Many hobbyists assume that anammox bacteria can only live in deep sand beds, however, they thrive in bacterial biofilms which can even be on the surface of live rock and at the surface of sand beds. These anaerobic areas are present where ever bacterial biofilms develop.
Keep in mind that a bacteria is very small, perhaps 0.5-10 microns, and a bacterial biofilm that has 30 layers of bacteria can easily have anaerobic areas. A bacterial biofilm that is 30 layers of bacteria high may only be 30 microns high. The human eye can only detect particles around 60 microns in size, so you may not be able to see biofilms that have anaerobic layers in it.
Bacteria do readily attach to sand particles found commonly in a reef aquarium. Bacterial biofilms can easily form around the surface of each sand particle further increasing the amount of anammox bacteria found in a reef aquarium. If nutrient levels are too high in a sand bed, the sand particles will start to glue together from the activity of these bioflims, which is a common occurance in sand beds according to posts made here on RC.
----------------------------------------------------------------------------------------------------
Copyright© 2007, American Society for Microbiology
In Situ Activity and Spatial Organization of Anaerobic Ammonium-Oxidizing (Anammox) Bacteria in Biofilms
Tomonori Kindaichi,1 Ikuo Tsushima,2 Yuji Ogasawara,2 Masaki Shimokawa,2 Noriatsu Ozaki,1 Hisashi Satoh,2 and Satoshi Okabe2
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1951037/
Abstract
We investigated autotrophic anaerobic ammonium-oxidizing (anammox) biofilms for their spatial organization, community composition, and in situ activities by using molecular biological techniques combined with microelectrodes. Results of phylogenetic analysis and fluorescence in situ hybridization (FISH) revealed that "œBrocadia"-like anammox bacteria that hybridized with the Amx820 probe dominated, with 60 to 92% of total bacteria in the upper part (<1,000 μm) of the biofilm, where high anammox activity was mainly detected with microelectrodes. The relative abundance of anammox bacteria decreased along the flow direction of the reactor. FISH results also indicated that Nitrosomonas-, Nitrosospira-, and Nitrosococcus-like aerobic ammonia-oxidizing bacteria (AOB) and Nitrospira-like nitrite-oxidizing bacteria (NOB) coexisted with anammox bacteria and accounted for 13 to 21% of total bacteria in the biofilms. Microelectrode measurements at three points along the anammox reactor revealed that the NH4+ and NO2− consumption rates decreased from 0.68 and 0.64 μmol cm−2 h−1 at P2 (the second port, 170 mm from the inlet port) to 0.30 and 0.35 μmol cm−2 h−1 at P3 (the third port, 205 mm from the inlet port), respectively. No anammox activity was detected at P4 (the fourth port, 240 mm from the inlet port), even though sufficient amounts of NH4+ and NO2− and a high abundance of anammox bacteria were still present. This result could be explained by the inhibitory effect of organic compounds derived from biomass decay and/or produced by anammox and coexisting bacteria in the upper parts of the biofilm and in the upstream part of the reactor. The anammox activities in the biofilm determined by microelectrodes reflected the overall reactor performance. The several groups of aerobic AOB lineages, Nitrospira-like NOB, and Betaproteobacteria coexisting in the anammox biofilm might consume a trace amount of O2 or organic compounds, which consequently established suitable microenvironments for anammox bacteria.
Keep in mind that a bacteria is very small, perhaps 0.5-10 microns, and a bacterial biofilm that has 30 layers of bacteria can easily have anaerobic areas. A bacterial biofilm that is 30 layers of bacteria high may only be 30 microns high. The human eye can only detect particles around 60 microns in size, so you may not be able to see biofilms that have anaerobic layers in it.
Bacteria do readily attach to sand particles found commonly in a reef aquarium. Bacterial biofilms can easily form around the surface of each sand particle further increasing the amount of anammox bacteria found in a reef aquarium. If nutrient levels are too high in a sand bed, the sand particles will start to glue together from the activity of these bioflims, which is a common occurance in sand beds according to posts made here on RC.
----------------------------------------------------------------------------------------------------
Copyright© 2007, American Society for Microbiology
In Situ Activity and Spatial Organization of Anaerobic Ammonium-Oxidizing (Anammox) Bacteria in Biofilms
Tomonori Kindaichi,1 Ikuo Tsushima,2 Yuji Ogasawara,2 Masaki Shimokawa,2 Noriatsu Ozaki,1 Hisashi Satoh,2 and Satoshi Okabe2
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1951037/
Abstract
We investigated autotrophic anaerobic ammonium-oxidizing (anammox) biofilms for their spatial organization, community composition, and in situ activities by using molecular biological techniques combined with microelectrodes. Results of phylogenetic analysis and fluorescence in situ hybridization (FISH) revealed that "œBrocadia"-like anammox bacteria that hybridized with the Amx820 probe dominated, with 60 to 92% of total bacteria in the upper part (<1,000 μm) of the biofilm, where high anammox activity was mainly detected with microelectrodes. The relative abundance of anammox bacteria decreased along the flow direction of the reactor. FISH results also indicated that Nitrosomonas-, Nitrosospira-, and Nitrosococcus-like aerobic ammonia-oxidizing bacteria (AOB) and Nitrospira-like nitrite-oxidizing bacteria (NOB) coexisted with anammox bacteria and accounted for 13 to 21% of total bacteria in the biofilms. Microelectrode measurements at three points along the anammox reactor revealed that the NH4+ and NO2− consumption rates decreased from 0.68 and 0.64 μmol cm−2 h−1 at P2 (the second port, 170 mm from the inlet port) to 0.30 and 0.35 μmol cm−2 h−1 at P3 (the third port, 205 mm from the inlet port), respectively. No anammox activity was detected at P4 (the fourth port, 240 mm from the inlet port), even though sufficient amounts of NH4+ and NO2− and a high abundance of anammox bacteria were still present. This result could be explained by the inhibitory effect of organic compounds derived from biomass decay and/or produced by anammox and coexisting bacteria in the upper parts of the biofilm and in the upstream part of the reactor. The anammox activities in the biofilm determined by microelectrodes reflected the overall reactor performance. The several groups of aerobic AOB lineages, Nitrospira-like NOB, and Betaproteobacteria coexisting in the anammox biofilm might consume a trace amount of O2 or organic compounds, which consequently established suitable microenvironments for anammox bacteria.
Last edited:
HighlandReefer
Team RC
Given the articles in this thread I have sighted and one article refereincing the fact that it is well known that ethanol inhibits anammox bacterial growth, what are the effects of dosing ethanol on the anammox bacterial populations in a reef aquarium & what are the consequences of this? I have not seen any studies about this in a reef aquarium (only studies regarding marine and fresh water waste reactors), true, but I can hypothesis about this.
HighlandReefer
Team RC
Anaerobic Ammonium-Oxidizing (Anammox) Bacteria and Associated Activity in Fixed-Film Biofilters of a Marine Recirculating Aquaculture System† (2006)
Yossi Tal1,
Joy E. M. Watts2 and
Harold J. Schreier1,3,*
+ Author Affiliations
1Center of Marine Biotechnology, University of Maryland Biotechnology Institute, 701 E. Pratt Street, Baltimore, Maryland 21202
2Department of Biological Sciences, Towson University, 8000 York Road, Towson, Maryland 21252
3Department of Biological Sciences, University of Maryland Baltimore County, 1000 Hilltop Circle, Baltimore, Maryland 21250
http://aem.asm.org/content/72/4/2896.abstract
ABSTRACT
Microbial communities in the biological filter and waste sludge compartments of a marine recirculating aquaculture system were examined to determine the presence and activity of anaerobic ammonium-oxidizing (anammox) bacteria. Community DNA was extracted from aerobic and anaerobic fixed-film biofilters and the anaerobic sludge waste collection tank and was analyzed by amplifying 16S rRNA genes by PCR using anammox-selective and universal GC-clamped primers. Separation of amplified PCR products by denaturing gradient gel electrophoresis and sequencing of the different phylotypes revealed a diverse biofilter microbial community. While Planctomycetales were found in all three communities, the anaerobic denitrifying biofilters contained one clone that exhibited high levels of sequence similarity to known anammox bacteria. Fluorescence in situ hybridization studies using an anammox-specific probe confirmed the presence of anammox Planctomycetales in the microbial biofilm from the denitrifying biofilters, and anammox activity was observed in these biofilters, as detected by the ability to simultaneously consume ammonia and nitrite. To our knowledge, this is the first identification of anammox-related sequences in a marine recirculating aquaculture filtration system, and our findings provide a foundation for incorporating this important pathway for complete nitrogen removal in such systems.
Yossi Tal1,
Joy E. M. Watts2 and
Harold J. Schreier1,3,*
+ Author Affiliations
1Center of Marine Biotechnology, University of Maryland Biotechnology Institute, 701 E. Pratt Street, Baltimore, Maryland 21202
2Department of Biological Sciences, Towson University, 8000 York Road, Towson, Maryland 21252
3Department of Biological Sciences, University of Maryland Baltimore County, 1000 Hilltop Circle, Baltimore, Maryland 21250
http://aem.asm.org/content/72/4/2896.abstract
ABSTRACT
Microbial communities in the biological filter and waste sludge compartments of a marine recirculating aquaculture system were examined to determine the presence and activity of anaerobic ammonium-oxidizing (anammox) bacteria. Community DNA was extracted from aerobic and anaerobic fixed-film biofilters and the anaerobic sludge waste collection tank and was analyzed by amplifying 16S rRNA genes by PCR using anammox-selective and universal GC-clamped primers. Separation of amplified PCR products by denaturing gradient gel electrophoresis and sequencing of the different phylotypes revealed a diverse biofilter microbial community. While Planctomycetales were found in all three communities, the anaerobic denitrifying biofilters contained one clone that exhibited high levels of sequence similarity to known anammox bacteria. Fluorescence in situ hybridization studies using an anammox-specific probe confirmed the presence of anammox Planctomycetales in the microbial biofilm from the denitrifying biofilters, and anammox activity was observed in these biofilters, as detected by the ability to simultaneously consume ammonia and nitrite. To our knowledge, this is the first identification of anammox-related sequences in a marine recirculating aquaculture filtration system, and our findings provide a foundation for incorporating this important pathway for complete nitrogen removal in such systems.
HighlandReefer
Team RC
I found the complete article noted above at this link:
Anaerobic ammonium-oxidizing (anammox) bacteria and associated activity in fixed-film biofilters of a marine recirculating aquaculture system.
http://ukpmc.ac.uk/articles/PMC1448996/reload=0;jsessionid=CF0E04208E12D7A5B0F91112E6C5EEFA
From it in part:
"Aquaculture biofilters and wastewater treatment environments.
With the exception of natural environments and wastewater treatment plants, there have been no reports of anammox-related Planctomycetales in water treatment systems that have characteristics of the organisms encountered in recirculating aquaculture systems. The important differences between water quality parameters of aquaculture systems and wastewater plants include the concentrations of nutrients and the organic loads. While the concentration of ammonia in raw sewage may reach 20 to 40 mg liter−1 (ammonia nitrogen, NH3-N) (51), the maximum ammonia concentrations allowed in aquaculture systems are less then 1 mg NH3-N liter−1, as higher concentrations of ammonia are lethal to fish (41). Moreover, the biological oxygen demand values for organic loads in raw sewage reach between 200 and 500 mg liter−1 (13), while they are usually less than 50 mg liter−1 in recirculating aquaculture systems (15). Furthermore, the growth structures of the bacterial communities are different. While activated sludge suspensions of wastewater treatment plants promote granule formation, a thin fixed film is the main structure of bacterial growth in moving bed biofilters of recirculating aquaculture systems. The ability of anammox bacteria to populate these distinct synthetic environments is consistent with the belief that the anammox process is amenable to many wastewater treatment applications with minimal apparent restrictions with respect to nutrient and salt concentrations, organic loads, or bacterial growth configurations (37).
Anammox versus denitrification.
Anaerobic incubation of beads from the denitrifying biofilter showed that the removal rates were 1.08 μM nitrate nitrogen bead−1 h−1 under denitrifying conditions and 0.098 μM ammonia nitrogen bead−1 h−1 and 0.16 μM hydrazine bead−1 h−1 under anammox conditions. Although the fraction of anammox bacteria in the consortia of the anammox-enriched beads was 12.5% ± 1.6%, as determined by FISH, the nitrogen removal rates were between 7- and 11-fold lower than the denitrification rates of untreated beads. This discrepancy was likely due to the low activity and growth rate (11 days) of the anammox bacteria and indicates that there is a major limitation in using the anammox process for nitrogen removal in these systems. Ammonia oxidation rates for cultures of anammox bacteria with nitrite as the electron acceptor have been determined to be 55 nmol min−1 mg protein−1 (16), compared to, for example, 400 nmol min−1 mg protein−1 for nitrate reduction by the heterotrophic denitrifyer Pseudomonas stutzeri (50). This more-than-sevenfold difference suggests that denitrifying bacteria may outcompete anammox bacteria, especially in an organic carbon-rich environment, and could explain the relatively low numbers of anammox bacteria in the denitrifying biofilter, which was populated predominantly by fast-growing heterotrophic denitrifiers (51). Recent studies have shown that anammox bacteria are capable of myxotrophic growth in the presence of fatty acids, such as acetate and propionate (14), which are common organic waste products of aquaculture filtration systems (49), supporting the notion that anammox and denitrifying bacteria can share the same environment. To further assess the specific contributions of anammox and denitrifying bacteria in the nitrogen removal process of the recirculating system will require the use of 15N-labeling experiments in which isotope pairing distinguishes between these two processes (16, 45).
Applying the anammox process.
Development of anammox as the major nitrogen removal process in recirculating aquaculture systems is advantageous due to the reduced oxygen demands and the autotrophic nature of the process, which allow complete nitrogen removal without a need for organic carbon. As noted by other workers (26, 46), the low growth rates and activity exhibited by anammox bacteria should be reflected in two major features of anammox-based biofilters, inoculation time and biomass retention efficiency. Long initiation periods under conditions favorable for anammox are required in order to establish a stable anammox population. Shortening this period may be possible by inoculation of lab-enriched anammox cultures, although the success of such an approach will depend largely on the type and origin of the anammox culture used. Inoculation of marine recirculating systems with anammox cultures that are already a component of the natural bacterial consortium of the anaerobic biofilters should improve the likelihood of success. High biomass retention efficiency of an anammox-based biofilter is essential to overcome the low activity of the anammox bacteria. High surface-to-volume ratios of the biofilter substrate should increase the anammox bacterial biomass and improve the overall activity per volume of biofilter.
Successful operation of an anammox biofilter requires a constant supply of ammonia and nitrite along with minimal input of organic carbon. Such an environment is necessary to maintain the anammox consortium, while reducing competition from denitrifying bacteria. One approach for enhancing anammox activity in the recirculating system used in this study would be to partially aerate the sludge tank to stimulate incomplete denitrification in order to promote nitrite accumulation. The nitrite-containing water would then be supplied to the anaerobic biofilter along with ammonia-rich wastewater from the fish tank. We are presently addressing whether this combination promotes growth and activity of the anammox bacteria in an anaerobic biofilter.
Conclusion.
Stricter environmental regulations for effluent discharge from aquaculture facilities are currently motivating the aquaculture industry to reduce effluent volumes and nitrogen loads. Closing the nitrogen cycle in the water treatment system by utilizing the anammox process is a desirable alternative to heterotrophic denitrification, especially in marine recirculating systems. Our finding that bacteria capable of anammox and their activity are associated with the consortia of a denitrifying biofilter makes this approach feasible for implementation and will direct future advances in building an environmentally sustainable land-based mariculture industry."
Anaerobic ammonium-oxidizing (anammox) bacteria and associated activity in fixed-film biofilters of a marine recirculating aquaculture system.
http://ukpmc.ac.uk/articles/PMC1448996/reload=0;jsessionid=CF0E04208E12D7A5B0F91112E6C5EEFA
From it in part:
"Aquaculture biofilters and wastewater treatment environments.
With the exception of natural environments and wastewater treatment plants, there have been no reports of anammox-related Planctomycetales in water treatment systems that have characteristics of the organisms encountered in recirculating aquaculture systems. The important differences between water quality parameters of aquaculture systems and wastewater plants include the concentrations of nutrients and the organic loads. While the concentration of ammonia in raw sewage may reach 20 to 40 mg liter−1 (ammonia nitrogen, NH3-N) (51), the maximum ammonia concentrations allowed in aquaculture systems are less then 1 mg NH3-N liter−1, as higher concentrations of ammonia are lethal to fish (41). Moreover, the biological oxygen demand values for organic loads in raw sewage reach between 200 and 500 mg liter−1 (13), while they are usually less than 50 mg liter−1 in recirculating aquaculture systems (15). Furthermore, the growth structures of the bacterial communities are different. While activated sludge suspensions of wastewater treatment plants promote granule formation, a thin fixed film is the main structure of bacterial growth in moving bed biofilters of recirculating aquaculture systems. The ability of anammox bacteria to populate these distinct synthetic environments is consistent with the belief that the anammox process is amenable to many wastewater treatment applications with minimal apparent restrictions with respect to nutrient and salt concentrations, organic loads, or bacterial growth configurations (37).
Anammox versus denitrification.
Anaerobic incubation of beads from the denitrifying biofilter showed that the removal rates were 1.08 μM nitrate nitrogen bead−1 h−1 under denitrifying conditions and 0.098 μM ammonia nitrogen bead−1 h−1 and 0.16 μM hydrazine bead−1 h−1 under anammox conditions. Although the fraction of anammox bacteria in the consortia of the anammox-enriched beads was 12.5% ± 1.6%, as determined by FISH, the nitrogen removal rates were between 7- and 11-fold lower than the denitrification rates of untreated beads. This discrepancy was likely due to the low activity and growth rate (11 days) of the anammox bacteria and indicates that there is a major limitation in using the anammox process for nitrogen removal in these systems. Ammonia oxidation rates for cultures of anammox bacteria with nitrite as the electron acceptor have been determined to be 55 nmol min−1 mg protein−1 (16), compared to, for example, 400 nmol min−1 mg protein−1 for nitrate reduction by the heterotrophic denitrifyer Pseudomonas stutzeri (50). This more-than-sevenfold difference suggests that denitrifying bacteria may outcompete anammox bacteria, especially in an organic carbon-rich environment, and could explain the relatively low numbers of anammox bacteria in the denitrifying biofilter, which was populated predominantly by fast-growing heterotrophic denitrifiers (51). Recent studies have shown that anammox bacteria are capable of myxotrophic growth in the presence of fatty acids, such as acetate and propionate (14), which are common organic waste products of aquaculture filtration systems (49), supporting the notion that anammox and denitrifying bacteria can share the same environment. To further assess the specific contributions of anammox and denitrifying bacteria in the nitrogen removal process of the recirculating system will require the use of 15N-labeling experiments in which isotope pairing distinguishes between these two processes (16, 45).
Applying the anammox process.
Development of anammox as the major nitrogen removal process in recirculating aquaculture systems is advantageous due to the reduced oxygen demands and the autotrophic nature of the process, which allow complete nitrogen removal without a need for organic carbon. As noted by other workers (26, 46), the low growth rates and activity exhibited by anammox bacteria should be reflected in two major features of anammox-based biofilters, inoculation time and biomass retention efficiency. Long initiation periods under conditions favorable for anammox are required in order to establish a stable anammox population. Shortening this period may be possible by inoculation of lab-enriched anammox cultures, although the success of such an approach will depend largely on the type and origin of the anammox culture used. Inoculation of marine recirculating systems with anammox cultures that are already a component of the natural bacterial consortium of the anaerobic biofilters should improve the likelihood of success. High biomass retention efficiency of an anammox-based biofilter is essential to overcome the low activity of the anammox bacteria. High surface-to-volume ratios of the biofilter substrate should increase the anammox bacterial biomass and improve the overall activity per volume of biofilter.
Successful operation of an anammox biofilter requires a constant supply of ammonia and nitrite along with minimal input of organic carbon. Such an environment is necessary to maintain the anammox consortium, while reducing competition from denitrifying bacteria. One approach for enhancing anammox activity in the recirculating system used in this study would be to partially aerate the sludge tank to stimulate incomplete denitrification in order to promote nitrite accumulation. The nitrite-containing water would then be supplied to the anaerobic biofilter along with ammonia-rich wastewater from the fish tank. We are presently addressing whether this combination promotes growth and activity of the anammox bacteria in an anaerobic biofilter.
Conclusion.
Stricter environmental regulations for effluent discharge from aquaculture facilities are currently motivating the aquaculture industry to reduce effluent volumes and nitrogen loads. Closing the nitrogen cycle in the water treatment system by utilizing the anammox process is a desirable alternative to heterotrophic denitrification, especially in marine recirculating systems. Our finding that bacteria capable of anammox and their activity are associated with the consortia of a denitrifying biofilter makes this approach feasible for implementation and will direct future advances in building an environmentally sustainable land-based mariculture industry."
HighlandReefer
Team RC
I have started investigating the effects of acetate on marine algae. Several articles have found that acetate increases growth of your calcerous algae which IMO is a good thing in many cases, especially some macroalgae and coralline algae.
As far as the effects of acetate on microalgae which can be considered a pest in many cases, I have found this article so far:
The effect of organic substrates
on the growth, photosynthesis and
dark survival of marine algae
Sean Coughlan a
a Department of Plant Sciences, Leeds University, Leeds, LS2
9JT
Available online: 17 Feb 2007
http://www.tandfonline.com/doi/pdf/10.1080/00071617700650171
From it:
"RESULTS
Table I shows the growth characteristics (length of lag phase and relative
growth rate) of the three algae for different concentrations of glycollate, glucose
and acetate at 20°C and low light intensity. Only in the case of Thalassiosira
pseudonana growing on acetate did there appear to be any observable differences
in growth rate, but a two-way analysis of variance revealed that the differences
between the control flask (mean generation time = 18.4 h) and the experimental
flasks (mean generation time = 13-3 h for 10 pg 1-1 acetate) were not significant.
In all cases no dark growth was observed. Under the experimental conditions
used, where there was adequate nutrient and inorganic carbon and a limiting
energy source for autotrophic growth, neither glycollate, glucose nor acetate at
a variety of concentrations affected the growth characteristics of the three algae."
As far as the effects of acetate on microalgae which can be considered a pest in many cases, I have found this article so far:
The effect of organic substrates
on the growth, photosynthesis and
dark survival of marine algae
Sean Coughlan a
a Department of Plant Sciences, Leeds University, Leeds, LS2
9JT
Available online: 17 Feb 2007
http://www.tandfonline.com/doi/pdf/10.1080/00071617700650171
From it:
"RESULTS
Table I shows the growth characteristics (length of lag phase and relative
growth rate) of the three algae for different concentrations of glycollate, glucose
and acetate at 20°C and low light intensity. Only in the case of Thalassiosira
pseudonana growing on acetate did there appear to be any observable differences
in growth rate, but a two-way analysis of variance revealed that the differences
between the control flask (mean generation time = 18.4 h) and the experimental
flasks (mean generation time = 13-3 h for 10 pg 1-1 acetate) were not significant.
In all cases no dark growth was observed. Under the experimental conditions
used, where there was adequate nutrient and inorganic carbon and a limiting
energy source for autotrophic growth, neither glycollate, glucose nor acetate at
a variety of concentrations affected the growth characteristics of the three algae."
HighlandReefer
Team RC
This article relates to fresh water algae (this is a stretch):
Physiological Responses of Phytoflagellates to Dissolved Organic Substrate Additions. 1. Dominant Role of Heterotrophic Nutrition in Poterioochromonas malhamensis (Chrysophyceae)
Alan J. Lewitus1 and
David A. Caron
+ Author Affiliations
Biology Department, Woods Hole Oceanographic Institution, Woods Hole Massachusetts 02543, U.S.A.
1 Current address and address for correspondences: Horn Point Environmental Laboratories, University of Maryland, PO Box 775, Cambridge, Maryland 21613, U.S.A.
Received October 29, 1990.
Accepted May 8, 1991.
Abstract
Algal heterotrophy is a potentially important consideration in the flow of carbon through aquatic food webs. The physiological responses to organic compound additions under various light intensities were examined with Poterioochromonas malhamensis, a freshwater chrysophyte with an exceptionally high heterotrophic capability. P. malhamensis demonstrated a much greater potential for heterotrophic growth than for photoautotrophic growth. When organic substrates (glucose, glycerol, or ethanol) were added to the culture medium, the growth rate of P. malhamensis significantly increased while the chlorophyll α content cell −1 decreased, even at light intensities saturating for photoautotrophic growth. After an initial decline in chlorophyll production caused by organic substrate uptake, chlorophyll α cell1 increased and the uptake rate of organic substrates decreased, despite the persistence of a relatively high substrate concentration in the medium. The results are consistent with the production of substance(s) by P. malhamensis that conditioned the culture medium, leading to a relief of the inhibitory effect of organic substrates on chlorophyll production.
Physiological Responses of Phytoflagellates to Dissolved Organic Substrate Additions. 1. Dominant Role of Heterotrophic Nutrition in Poterioochromonas malhamensis (Chrysophyceae)
Alan J. Lewitus1 and
David A. Caron
+ Author Affiliations
Biology Department, Woods Hole Oceanographic Institution, Woods Hole Massachusetts 02543, U.S.A.
1 Current address and address for correspondences: Horn Point Environmental Laboratories, University of Maryland, PO Box 775, Cambridge, Maryland 21613, U.S.A.
Received October 29, 1990.
Accepted May 8, 1991.
Abstract
Algal heterotrophy is a potentially important consideration in the flow of carbon through aquatic food webs. The physiological responses to organic compound additions under various light intensities were examined with Poterioochromonas malhamensis, a freshwater chrysophyte with an exceptionally high heterotrophic capability. P. malhamensis demonstrated a much greater potential for heterotrophic growth than for photoautotrophic growth. When organic substrates (glucose, glycerol, or ethanol) were added to the culture medium, the growth rate of P. malhamensis significantly increased while the chlorophyll α content cell −1 decreased, even at light intensities saturating for photoautotrophic growth. After an initial decline in chlorophyll production caused by organic substrate uptake, chlorophyll α cell1 increased and the uptake rate of organic substrates decreased, despite the persistence of a relatively high substrate concentration in the medium. The results are consistent with the production of substance(s) by P. malhamensis that conditioned the culture medium, leading to a relief of the inhibitory effect of organic substrates on chlorophyll production.
HighlandReefer
Team RC
Provided your evaporation rate is normal, adding 15 ml of vinegar per 1 gallon of rodi water for top-off is a good starting point IME. I currently add 45 ml of vinegar per gallon without problems.
Keep in mind, if you add more than 2 teaspoons of kalk per 1 gallon of rodi water, the vinegar will dissolve the extra kalk and increase your output of alk and calcium. At 3 teaspoons kalk per 45 ml of vinegar per 1 gallon of rodi water you may get 30-50% more alk and calcium output.
I believe Randy discusses the use of vinegar in kalk water in this article (the links are currently down to this article):
What Your Grandmother Never Told You About Lime
http://reefkeeping.com/issues/2005-01/rhf/index.htm
Keep in mind, if you add more than 2 teaspoons of kalk per 1 gallon of rodi water, the vinegar will dissolve the extra kalk and increase your output of alk and calcium. At 3 teaspoons kalk per 45 ml of vinegar per 1 gallon of rodi water you may get 30-50% more alk and calcium output.
I believe Randy discusses the use of vinegar in kalk water in this article (the links are currently down to this article):
What Your Grandmother Never Told You About Lime
http://reefkeeping.com/issues/2005-01/rhf/index.htm
Last edited:
HighlandReefer
Team RC
The pH of kalk water is high perhaps around 12.5. Adding vinegar to kalk water will reduce the pH of kalk water a bit, but not that much. So the overall effect is that kalk water with vinegar added will still increase your tank pH.
Provided your evaporation rate is normal, adding 15 ml of vinegar per 1 gallon of rodi water for top-off is a good starting point IME. I currently add 45 ml of vinegar per gallon without problems.
Keep in mind, if you add more than 2 teaspoons of kalk per 1 gallon of rodi water, the vinegar will dissolve the extra kalk and increase your output of alk and calcium. At 3 teaspoons kalk per 45 ml of vinegar per 1 gallon of rodi water you may get 30-50% more alk and calcium output.
I believe Randy discusses the use of vinegar in kalk water in this article (the links are currently down to this article):
What Your Grandmother Never Told You About Lime
http://reefkeeping.com/issues/2005-01/rhf/index.htm
Thank you
alessandro
New member
Someone may also find useful to replace vinegar with GAA (glacial acetic acid).
GAA is 20x more powerful than vinegar, i.e. 20 ml. of vinegar is equivalent to 1 ml of GAA.
GAA is 20x more powerful than vinegar, i.e. 20 ml. of vinegar is equivalent to 1 ml of GAA.
Telaverus
New member
Is it better to use vinegar as it is regulated and required to be made from organic sources, whereas GAA can be made from a petrolium source?
Does it matter?
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