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How to Control Nitrite in Biofloc Systems

 

Gilbert Thiriez (Email acuaguajaro@yahoo.com), a shrimp farmer in Colombia, reports to The Shrimp List:

 

I’m working on a high-density, pilot, biofloc shrimp farm.  My nitrite levels are too high, and I’m getting some mortalities and soft shells.  I have tried increasing the water exchange to 8%, but the high nitrite levels don’t go away.

 

Here are my pond specifications:

 

Stocking Density = 300 shrimp per square meter (m2)

Pond Size = 500 square meters (m2)

Pond Depth = 1.5 meters

Biomass = about 790 kilograms per pond (15.8 tons/hectare)

Aeration = 60 horsepower per hectare

Paddlewheels = edge of the pond

Air Injectors = center of the pond

Water Quality = dissolved oxygen 4.3 milligrams per liter (mg/l)

Temperature = 29.5°C

Salinity = 48 parts per thousands

pH  = 7.1

Alkalinity = 400 mg/l CaCO3

Ammonia  = 0.006 mg/l

Nitrite = 61.2 mg/l

Nitrate = 89.9 mg/l

Phosphates = 32 mg/l

Bottom Quality = No black spots

Feed = Nicovita 35% protein

Carbon Source = Molasses

Pond Management = Drain from the center of the pond every day

Water Exchange/Day = 2%

 

Please forward some ideas for controlling and lowering nitrite levels.

 

Jack Crockett (Email hondurasjack@yahoo.com): You can control nitrite levels by organic carbon addition.  Any carbon you add is first used by heterotrophic bacteria in combination with ammonia to build protein.  If there is an excess of organic carbon, bacteria next use nitrites as a nitrogen source to build protein.  It takes a little longer to reduce nitrite with organic carbon because first the bacteria need to reduce nitrites to ammonia by enzymatic action, but any form of inorganic nitrogen can be controlled by addition of organic carbon.  If you want more information on how to calculate the amount of organic carbon needed, please email me.

 

Dr. Abraham Joseph (Email microbioster@gmail.com):  You should  consider using feed with 25-30% protein, instead of 35% protein, because the nitrogenous metabolites like NH3, NO3 and NO2 are derived from protein metabolism.  As you know, biofloc materials are eaten by shrimp and may reduce the feeding requirement by 25%.  Biofloc is a good source of microbial protein (single cell protein).  Try to increase the carbon level by adding more molasses at regular intervals.  Keep the oxygen the same as you listed above.  You may also consider a top quality functional probiotic with synergistic species of bacteria that can utilize nitrogen-based metabolites to reduce the NO2 level.

 

Your C:N:P (carbon:nitrogen:phosphate) ratio should be 100:5:1.  Your biofloc system is deficient in carbon and too high in nitrogen and phosphate.  Follow a carbon chart and add adequate molasses to fix the NO2 issue.  Again, NO2 is toxic at high concentrations and you must take immediate steps to lower it.

 

Eric De Muylder (Email eric@crevetec.be): Is your nitrite 60 parts per million (ppm) NO2 or 60 ppm NO2-N.  If it is 60 ppm NO2, it corresponds with about 20 ppm NO2-N.  Although it is high, it is  not critical, especially at 48 ppt salinity.

 

If you add more molasses or other carbon, you will need to add 200-ppm carbon.  If you do that, your oxygen will drop and your biofloc density will explode.  You might solve your problem in the short term, but then face a lot of other issues.  What is your biofloc density now?  Give it to me in milligrams per liter from an Imhoff cone.

 

In the short term, you need to cut down on feed and stop using molasses.  Overfeeding could be part of the problem.  You also need to use more aeration.  If you reduce the amount of molasses that you are using, your oxygen level will automatically increase.

 

Yes, your soft-shell problem could be caused from high levels of nitrite, but it also could be from carbon dioxide (CO2 ) or from a mineral imbalance.  Why is your salinity so high?  Is your water source that high, or does it increase with evaporation?  If it’s caused by evaporation, a mineral imbalance could be your problem.  But if you are exchanging a lot of water, that shouldn’t be your problem.

 

I realize my mail is not really helping you.  I fear your system is not appropriate for biofloc farming.

 

The main problem is that you don’t have enough nitrifying bacteria to convert the nitrite to nitrate.  It usually takes a long time to establish enough nitrifying bacteria.  Do you re-use your pond water after harvesting, or do you start from zero on every crop?

 

Gilbert Thiriez (Email acuaguajaro@yahoo.com), who started this discussion on nitrites, says we believe that a possible cause of our problem is that the pH is too low to affect the growth of Nitrobacters.  Below he responds to Eric De Muylder’s questions (above):

 

What is your biofloc density?  Imhoff cone: 13 ml, before an 8% water exchange, it was in 20–30 ml.

 

Why is your salinity so high?  The high salinity is due to evaporation during the dry season.

 

Do you re-use your pond water after harvesting, or do you start from zero after every crop?  We re-use 1/3 of the pond water after harvesting.

 

Diego Maia Rocha (Email diegomaia@aquatec.com.br): Gilbert, How long is your cycle?  How are your stocking (directly, single phase or two phase)?

 

Tzachi Samocha (Email t-samocha@tamu.edu): Based on our experience, to keep the ammonia and nitrite under control (1 to 2 mg/l) you should try to reach the stage where these metabolites are being processed by the nitrifying bacteria.

 

Your description suggests that currently your system is heterotrophically dominated, meaning most of the ammonia is being processed by the heterotrophic bacteria.  Our experience shows that under no exchange, establishing a healthy nitrifying bacteria in your tank, will keep ammonia and nitrite at very low levels (1-2 mg/l) even when shrimp biomass load is high (> 9kg/m3) with no supplementation of organic carbon.

 

I suggest you start to develop these nitrifying bacteria in your tank as quickly as you can.

 

If you have access to commercial nitrifying bacteria concentrate, you may want to add it to your tank to accelerate the development of nitrifying bacteria in your system.  Make sure you select the product designed to work in seawater rather than freshwater systems.  During this process you will need to follow very closely the changes in ammonia, nitrite, nitrate and alkalinity to determine the rate of development of your nitrifying bacteria.  Your objective should be to reduce the daily applications of organic carbon to zero (the stage when nitrifying bacteria can process all of the ammonia left after the heterotrophic bacteria assimilated the ammonia in the system using organic carbon from feed or decaying organic material in your system).

 

Ramon Macaraig (Email monmac52@yahoo.com): We are also testing a biofloc system in open ponds.  After 116 days of growout, we harvested 2,070 kilograms of 20-gram shrimp from 1,200 m2 ponds that are 1.2 meters deep.  Stocking density was 160 PL/m2.  Final weights averaged 23 grams, with one 400-kilogram partial harvest at 17 grams.  Our survival rate was 50%.  The floc density stabilized at 12 cm per liter of pond water, but it took a while to establish.  The early morning oxygen level never went below 5 ppm.  Temperature was 28-31°C, salinity was 20 ppt, pH ranged from 7.5-8.0.

 

We were not aware of the use of alkalinity and had chronic soft shell problems towards the end.  It was minimized by adding 300 kilograms of calcitic lime per hectare per week.

 

On ammonia levels, we work on the principle that biofloc systems will develop when oxygen levels are more than 10 ppm, which in a pond can be induced by algae growth in the morning.  We allow the green algae to have first crack at the nitrogenous waste, and allow it to bloom till 25-30 cm by noontime.  That’s when the oxygen levels gets higher than 10 ppm.  Airlift aeration and paddlewheels at a total of 40 horsepower per hectare keep the floc suspended.  We measure the total ammonium nitrate (TAN) and add molasses at 20 times the volume of the TAN.  We do this at least twice a week just to maintain the Imhoff floc volume.  Feeds of 35% are used to maintained the floc density.

 

Our crew said the shrimp were fat and very sweet, though their shells were rather thin.

 

We are stocking three more 1,200 m2 ponds at 160-200 PLs this week, and we will track the alkalinity this time after adding 300 kg of calcitic agricultural lime weekly.  These ponds are not lined with plastic, but use the same combination of paddlewheels and blowers as mentioned above.

 

I know our ponds have whitespot problems, but we hope that if we can get the morning oxygen up and the nitrogenous stressors from feed residues down, there is a chance to improve survivals, raise the carrying capacity, extend the growout period, produce 30-count P. vannamei and make money.

 

Dallas Weaver (Email deweaver@me.com):  Gilbert, after looking at the your water chemistry, I see a lot of things that don’t make sense.

 

The high alkalinity in 8 meq/l range is inconsistent with a pH of 7.1 and any reasonable “free CO2” (CO2 partial pressure in the water) concentration that wouldn’t be harming the shrimp.  More than likely, this is just a total alkalinity measurement in a water containing significant amounts of soluble, ionizable organic acids for biological materials (organic buffers not carbonates) and very little real “carbonate alkalinity”.  You need some carbonate alkalinity to get proper nitrification reactions from ammonia all the way to nitrate. 

 

You can check for the carbonate alkalinity by looking at the “equilibrium pH”.  Just take a sample and aerate the sample with an air stone using air from outside (no building air) which has about 400 ppmv of CO2.  Aerate long enough for the pH to come to equilibrium where the CO2 partial pressure in the water is the same as the air (this may take several hours).  If the pH doesn’t come up to the high 7s to low 8s, you don’t have enough carbonate alkalinity.  If that is the case, you may want to add (slowly) some alkalinity. 

 

You report very high nitrate (as NO3 (N) or as NO3?).  In the presence of high nitrite, nitrate test kits and colorimetric reading can give false results.  Make up some test standards with NO2 contamination and find out.  You may just have lots of nitrite and very little nitrate. 

 

The high levels of nitrite (as (N) or NO2?) does indicate nitrification bacteria in the system, which is good, however you need to grow more of the bacteria that go from nitrite to nitrate.  If you can get a lot of your nitrogen in the form of nitrate, it will suppress H2S formation in bottom sediments (beneficial—especially for systems prone to sludge deposits with bioflocs).

 

However, both of the bacteria species that go from ammonia to nitrite and from nitrite to nitrate are slow growing (as bacteria go) and can be out competed by heterotrophic bacteria using sugars.  Your shift from 2% exchange a day, which allows a SRT (sludge retention time—average biofloc age ) greater than 14 days, will allow nitrification bacteria to become common in the floc.  However, if that ammonia level of 0.006 is actual total ammonia N (TAN) (how did you measure that low if it is TAN?), there is not enough TAN to grow nitrification bacteria and all the ammonia is just becoming molasses based heterotrophic biomass.  If it is unionized ammonia, that is something else? 

 

Going to 8% a day exchange will almost flush out any nitrifying bacteria you have, especially with heterotrophic competition and low ammonia levels.  This is the unstable rate of exchange for nitrification and the only method of handling the ammonia buildup at 8% is to add even more carbon to create biomass using the nitrite and flush out 8% of that biomass every day and not be able to drop the 8% water rate back to 2% without a big increase in suspended solids.  You will also be using a lot of molasses with a huge oxygen demand (hope you have enough aeration capacity).

 

I have always wanted to do an experiment using a small secondary pond, where you fill it with some of your high nitrite water but no animals and lots of aeration and mixing.  I would then add enough urea or other ammonia source to the pond water to maintain about 6 mg/l of TAN (total ammonia N).  This small pond should, within a month or so, convert ammonia (being fed ammonia daily to maintain about 6 mg/l) and nitrite to nitrate as the biofloc becomes dominated by these slow growing nitrifying bacteria.  The biofloc from this pond probably has no shrimp pathogens (no shrimp hosts) and could be used as bacteria seed stock for supplementing and stabilizing a pond like the one described with too much nitrite and not enough nitrite to nitrate bacteria.  You could, in theory with continuous ammonia feed, maintain a stable population at 5% harvest of active biomass a day to help a pond in trouble.

 

The mathematics and models work.

 

PS: the phosphate seems high (as P or PO4, I like mmol/l which gets around this chemical species issue).  That may indicate that your calcium is too low and you have no fluoride in the water (just a guess).  If so, I would add some Ca(OH) PO2 very slowly as a highly dispersed slurry to increase the Ca and carbonate alkalinity. 

 

Brian Boudreau (Email brianboudreau01@yahoo.com): Gilbert, you can grow a special nitrifying bacteria at a very highly concentrated level in a smaller tank ideally around 20m3 for a 500 m3 production tank, then inoculate into the growout tank.  These bacteria are capable of dropping NO2 levels from 30 to 0.1mg/L overnight in the 20 m3 tank.  You will need lots of nitrite and bicarbonate.

 

The way to do it is with a lot of aeration and what I call successive pulsing to develop and accumulate this nitrite consuming bacteria.  With a high nitrite level like yours, start by filling the 20 m3 tank with high nitrite water (60mg/L) and add bicarbonate and aeration until nitrite levels are reduced to less than 1.  Then stop aeration and let the floc fall out of the water column and drain off the water above the floc with a submersible pump, being careful not to lower the pump to floc level.  Fill again with high nitrite water from growout and repeat.  Each time, a greater amount of nitrite floc will develop and accumulate.  You will see exponential increases in floc concentration and nitrite consumption.  Then start dosing 500-m3 growout tanks with this inoculate, leaving half of the culture to continue culture in the smaller tank (20 m3).  Keep this concentrated nitrifying bacteria going.

 

David Paladines C. (Email dfpaladines@hotmail.com): Hello Gilbert, I’ve been researching these bacteria for over ten years and as Dr. Samocha (above) says they are very effective at converting ammonia and nitrite to nitrate.  In trials I’ve seen them nitrify up to 100 ppm TAN a day in pure culture.  I can help you with these type of bacteria.

 

Dallas, a long time ago, I did the experiment you mention (above), and I can tell you it works!  You can do that or add a nitrifying bacteria concentrate, which speeds nitrification and you start reading nitrate levels the first days, and you won’t have TAN and nitrite spikes.

 

Dr. Mark Rigby (Email mrigby@llyn-aquaculture.co.uk): Gilbert, we have found—I think it is well documented—that the Nitrobacter group of bacteria are far more sensitive that the Nitrosomonas group.  Hence any “upset”’ in your system will cause rapid nitrite spikes while ammonia remains low.  This can be temperature, oxygen, pH or salinity—or combination of these, plus lack of alkalinity.  Your salinity at 48 ppt is really high, and I would expect it to kill everything!  If you’re outside, how does heavy rain (or lack of rain ) affect your water quality?

 

Working indoors with low light, we’ve gone up to 8 kg/m2 without the use of additional carbon, but do need to use pure oxygen.

 

If you’re working in outdoor ponds, I guess you have a lot of phytoplankton in the ponds as well, which  complicates the issue.  Do you see big diurnal fluctuations in water parameters?  This could be killing off your friendly Nitrobacters!  They are particularly sensitive to rapid pH swings in our experience.  At the densities we’ve used, we have to use sodium hydroxide and on occasions when a dosing pump has blocked the first thing we see the next day (other than a drop in pH) is a rise in NO2.

 

Source: The Shrimp List (a mailing list for shrimp farmers).  Subjects: a. How to Control High Nitrite Levels in Intensive Shrimp Biofloc System, b. Control of High Tan and Nitrite Levels in Intensive Shrimp Biofloc System, c. Control of TAN and Nitrite.  April 22–25, 2016.  2. Bob Rosenberry, Shrimp News International, April 25, 2016.

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