Probiotic bacterial cultures added to shrimp ponds typically are composed primarily of heterotrophic bacteria or a mixture of heterotrophic bacteria and autotrophic nitrifiers. Heterotrophic bacteria are those bacteria that primarily obtain their nutrition from organic sources.  The primary source of carbon for these bacteria is carbohydrates.  Nitrogen is typically obtained from the proteins in the organic material consumed by the bacteria.  Just like the shrimp, heterotrophic bacteria excrete ammonia as a by-product of the metabolism of the proteins they consume.  Some heterotrophic bacteria, however, are able to utilize ammonia directly as an alternative source of nitrogen.




What does this all have to do with C: N ratios?  Shrimp feeds used in intensive shrimp ponds typically have at least 35% protein.  These feeds do not contain a lot of carbohydrates.  C: N ratios in these feeds typically run around 9:1.  The bacteria require about 20 units of carbon per unit of nitrogen assimilated.  With such a low C:N ratio in the feed, carbon is the limiting nutrient for heterotrophic bacteria populations.  The bacterial population will not expand beyond a certain point due to the limited availability of carbon.  The protein in the organic detritus supplies most of the nitrogen requirement for the heterotrophic bacteria under these circumstances, and inorganic ammonia is not utilized as a nitrogen source to any great extent.


If the C: N ratio is increased, either by feeding lower protein feeds with a higher percentage of carbohydrate, or by adding a carbohydrate source such as molasses in addition to the regular feed, the increased availability of carbon allows the heterotrophic bacterial population to consume a higher percentage of the protein in the organic material.  This results in a complete digestion of the organic material in the pond by the heterotrophic bacteria.  As the C: N ratio increases, the heterotrophic bacteria resort increasingly to ammonia metabolism to meet their nitrogen requirements.  As C: N ratios are increased even further, a point is reached where nitrogen, rather than carbon, becomes the limiting nutrient.  At this point, ammonia concentrations should be close to 0 mg/L in the pond.


It should be pointed out that holding the feed protein constant and supplementing with pure carbohydrate will result in much higher bacterial counts in the pond.  The oxygen required to support this additional bacterial biomass will increase proportionally with the increase in bacterial population.  Likewise, CO2 production will increase, driving pH down.  If you are contemplating carbohydrate supplementation to increase C: N ratios, make sure that your pond is well-aerated and circulated to keep the organic detritus suspended in the water column where there is sufficient oxygen for the heterotrophs.  Also, once you develop a dense population of heterotrophs through carbohydrate supplementation, don’t discontinue the carbohydrate supplementation suddenly.  This will starve the bacteria of carbon, a die-off will occur and you will get an ammonia spike.




Another point that should be considered before enhancing C: N ratios in P. monodon ponds.  P. monodon does not utilize the organic detritus and associated bacterial protein as effectively as a food source as does P. vannamei.  With vannamei, C: N ratios can be enhanced by lowering the overall feed protein levels and utilizing feeds that are high in carbohydrate.  Because vannamei feeds on the organic flocs and utilizes bacterial protein efficiently, growth rates don’t suffer and protein utilization efficiencies improve dramatically.  With monodon, feeding low-protein, high-carbohydrate diets will likely result in lower growth rates.  Therefore it might be necessary to rely more on supplementation with pure carbohydrates to boost C: N ratios.  But this will result in more bacterial biomass, more BOD, and higher CO2.  This makes it somewhat questionable, in my mind, whether it is worth the risk to manage a monodon pond with high C: N ratios.


Most common genera of heterotrophic bacteria used in probiotic formulations are Bacillus and Lactobacillus, both of which are gram-positive.  It is not necessary, however, to inoculate a pond with commercial probiotics in order to manage a heterotrophic production system.  This can be accomplished simply by maintaining a C:N ratio greater than 12:1, and supplying adequate aeration.  The bacteria are already present in every pond.  By removing the carbon (and perhaps oxygen) limitation, they will proliferate.


The counts of naturally occurring bacteria are several thousand per milliliter, so a one-hectare pond contains astronomical amounts of bacteria.  It would be very difficult to add enough bacteria to a pond to significantly change its bacterial composition.  


Also, one might expect that the naturally occurring bacteria species are the best adapted to the conditions in the pond.  There is no guarantee that the bacteria in the probiotic culture will be well adapted to the conditions in the pond, let alone that they will out-compete the naturally occurring bacteria species.  Even if enough bacteria were added to have an effect on bacterial composition at one point in time, it would likely be necessary to re-inoculate the bacteria periodically to maintain the predominance of the probiotic species.  I admit that there have been studies which appear to show benefits in terms of survival in probiotic-treated ponds.  But there are also a lot of studies that fail to find any measurable impact on bacterial species composition.  Perhaps there is something going on that enables the probiotic bacteria to positively influence survival even when they are not the predominant species.


Bacillus and Lactobacillus are common genera of heterotrophic bacteria used in probiotic formulas.  What genera of the heterotrophic bacteria are already in the ponds, but not in the commercial probiotic products?


Marine soil sediments contain naturally occurring beneficial bacteria such as Bacillus subtillis,B. circulans, B. megaterium, B. polymyxa, and B. licheniformis.  They are purified and multiplied in fermenters and then further processed as liquids or spray-dried powders for marketing in vegetative or spore forms).




Also, what’s the best way to measure the C: N ratio in a pond? 


Measurement of C/N is only part of the story.  If you measure TOC (total organic carbon), some of that carbon can be refractory and not help grow bacteria and soak up the ammonia.  Measuring TOC and BOD (biological oxygen demand, with and without ammonia oxidation inhibition) along with TKN (total Kjeldahl nitrogen) will provide some useful management information.  To make these systems work, you should also be rearing a species that can use the single-cell protein being produced in the pond.  If not, all you are doing is converting ammonia into an unusable biomass using a significant amount of carbohydrate and oxygen.  You either have to discharge that biomass or oxidize it in the pond bottom when drained.  If it stays in the system, it will metabolize itself back into ammonia and CO2. 


The only difference between a photosynthetic system (algae in a pond) and a heterotrophic system (carbohydrate and oxygen) is the energy supply for the waste treatment function.  Sunlight limits your energy density per unit area in algae-based systems, which limits your feed/area.  With heterotrophic systems the energy density is not limited; it’s volumetric. 


The real trick is to get the biomass from these waste systems into a usable animal as fast and efficiently as possible so you don’t waste energy redoing the ammonia again and again as the biomass (or algae) you produced with your energy input decays.  


Remember: all closed aquaculture is polyculture.  The only question is how many sellable species do you have and what are your energy flows.  The job of an aquaculturist is to control that microbiological ecology to get the energy flows and treatment biomass to go where you want.