Pond bacterial processes.

General bacterial processes in the pond

Bacteria work in the pond filling all the tiny ecological niches for which they are adapted. Bacteria are almost everywhere, and the different types have traits that allow them to grow in even the harshest conditions. In aquaculture ponds, bacterial processes have profound effects on the water quality, and it is useful to have some understanding of these processes.

Pond bacteria decompose organic material. The organic material comes from the applied food or from autotrophic activities of algae and cyanobacteria.

The way the bacteria decompose organic material affects the pond. This is because the products of decomposition may cause events in the pond due to their nutritive value or to their toxicity.

In general terms waste in ponds goes through the following stages of decomposition:

• Waste (uneaten feed, prawn/fish/zooplankton faeces, dead algae) is initially broken down to smaller molecular weight units by hydrolysing bacteria. These smaller units are then decomposed either aerobically or anaerobically.

• In aerobic decomposition, bacteria use oxygen to decompose the organic waste. The end products are CO₂ and bacterial cell mass.

• In anaerobic decomposition in marine ponds, a multi step process involving fermentation then sulphate reduction results in the end product hydrogen sulphide.

These processes are discussed in more detail below.

Hydrolysis

Hydrolysis is the first step in the breakdown of organic material in the pond. For example, when a bloom "crashes" a range of bacterial and fungal hydrolytic enzymes initiate the breakdown of the cell walls and release the cell contents.

The initial breakdown of dead algae, cyanobacteria and other high molecular weight organic material in the pond is the slowest step in the decomposition process. If it could be speeded up it would be very useful, as it would reduce the accumulation of sludge and its associated problems. Addition of very large numbers of active "probiotic", enzyme active bacteria into the pond is one way that seems to help speed things up. Maintaining a level of dissolved organic material in the water column may also be useful as it helps to maintain a foundation population of active enzyme active heterotrophs in the water column.

Aerobic decomposition

This is the growth of bacterial cells using organic carbon as the source of energy for growth, and oxygen as the electron acceptor. This is the most efficient decomposition process when a well balanced C:N:P ratio and plenty of oxygen are present. If organic C is in short supply, ammonia and other partially degraded decomposition products are released during the decomposition process. The result of C limitation is inefficient microbial degradation and subsequent accumulation of sludge. Excess C can also be a problem, slowing the whole process down as the bacteria need to oxidize the excess C until the optimum C:N ratio is reached.

If oxygen is lacking, DO levels can reduce dramatically and kill the prawns and fish. In aquaculture ponds, organic C is often needed to balance the equation because most of the material being decomposed is protein based. Molasses is useful here as it helps to balance the C:N. Care is required when adding molasses to ensure that oxygen levels can be maintained.

Fermentation

Fermentation is a process carried out by bacteria and fungi which breaks down organic material when no oxygen is present. In the pond, fermentation occurs in the "dead areas" where organic material tends to collect due to lack of water flow in those areas. The products of fermentation can be smelly intermediate by products that give the water a distinctive bad odour.

Fermentation is generally a slow process when compared to aerobic decomposition. Many microbes have the ability to switch between fermentation and aerobic growth (so called “facultative anaerobes”). When growing anaerobically, these fermenters are inefficient at reproducing themselves but are able to break down organic material to intermediate end products.

These intermediate end products are often smelly and sulphidous and have a detrimental impact on water and pond bottom quality. The process of partial breakdown of proteinaceous organic material by fermentation is often called putrefaction due to the bad smell. H₂S can be produced in particular if the waste material has a low C:N ratio.

In order to speed up the processes involved in anaerobic decomposition of waste organic material in the pond, organic carbon with a high C:N ration (molasses) is of tremendous benefit.

In summary, in marine pond management the objective is to speed up the aerobic “composting” of waste. Maintaining a good C:N ratio with addition of extra organic C and also maintaining good mixing and aeration is essential to achieve this goal. In most ponds however there will always be areas where some organic waste collects. In order to decompose this waste quickly and avoid accumulation, added C is beneficial as it minimises putrefaction and helps speed up the activity of the SRB’s (sulphate reducing bacteria) to mop up the fermentation byproducts.

Sulphate reduction

When bacteria work to decompose or "oxidise" organic material, they need to chemically "reduce" another chemical in order to complete the reaction.

When oxygen is present, it is the preferred reducing agent: oxygen is reduced to CO₂. When oxygen runs in short supply, the bacteria can utilise other molecules such as manganese, iron (Fe³⁺) or nitrate. When these are used up, sulphate (SO₄^3-) can be used by certain types of "sulphate reducing bacteria". When these SRB's use sulphate, they produce sulphide, which can be toxic to pond organisms, particularly fish and prawns. There are many different types of SRB that are able to use a range of organic substrates as their energy source. The organic substrates that SRB’s use are the products of fermentations that occur in the anoxic sludge. These organic products of fermentations include acetate and hydrogen gas as well as other compounds.

In ponds, the objective is to set the pond up so that there are no "dead areas" where organic material can collect. However, this can be difficult in practice, and usually there are some "dead areas" in the pond.

In these dead areas, organic material collects due to the water currents and as oxygen is rapidly used up, bacterial fermentation begins. These fermentations produce partially broken down organic by-products that are used by the SRB's (acetate, hydrogen gas etc). Addition of organic C has been shown to greatly speed up this activity and this is advantageous as it keeps the total amount of organic waste to a minimum in the pond.

The sulphate concentration is seawater is about 2,700 mg per litre, so salt water ponds are very susceptible to rapid growth of SRB's. The dead areas become sulphide "factories", pumping toxic sulphide into the water. Two common SRB process are shown below:

CH₃COO^- + SO₄^- + 3H⁺ -----> 2CO₂ + H₂S + 2H₂O

4H₂ + SO₄^- -----> H₂S + 2H₂O + 2OH^-

Both acetate and hydrogen are common products of fermentation of organic material and are also common substrates for the growth of SRB's. Note that the reactions produces CO₂ as well as H₂S and uses up H protons thereby causing an increase in TA.

If the C:N ratio is low (eg protein wastes) putrefaction products will accumulate and water quality will suffer.

Production of sulphide from these areas can often be accompanied by a drop in DO and a resultant drop in pH as CO₂ is pumped into the water by the active bacteria. When these all happen together, great caution is required to avoid stressing or even losing fish or prawns. pH can be raised to minimise the proportion of H₂S or better, peroxide can be applied to convert the H₂S to S.

Denitrification

This is the bacterial mediated process whereby nitrate (NO₃) is used by bacteria instead of oxygen when oxygen levels are low.

The denitrification process is a two step process, with nitrite produced as an intermediate:

Step 1: 6NO₃^- + 2CH₃OH -----> 6NO₂ + 2CO₂ + 4H₂O

Step 2: 6NO₂ + 3 CH₃OH -----> 3N₂ + 3CO₂ + 3H₂O + 6OH^-

These equations show that the 2nd step requires more carbon source than the first, so that in the event carbon is lacking, as is usually the case in ponds, NO₂ can accumulate. They also show that if enough C is present then the bioreaction can complete step 2 and release N gas back to the atmosphere.

The problem with denitrification is that it works best when oxygen is less than about 1 ppm: i.e. when it is low enough to severely stress or kill fish or prawns. This happens in sludge, so we must work hard to minimise sludge. Denitrification may happen within floc particles, the "insides" of which may have micro-zones with low DO. However in general terms, denitrification is probably not the ideal means to remove excess N within a prawn pond. External ponds for water recycling may however be useful, as DO's can be allowed to drop and allow denitrification to proceed.

Nitrification

Nitrification is the bacterial mediated conversion of ammonia to nitrite, and of nitrite to nitrate. These are two separate processes carried out by two different groups of bacteria.

The conversion of ammonia to nitrite is carried out by Nitrosomonas spp. as well as some other related species of bacteria. When these bacteria use the ammonia they assimilate the N from the ammonia and create more bacterial cells, as shown here:

55NH₄⁺ + 5CO₂ + 70O₂ -----> C₅H₇O₂N + 54NO₂ + 52H₂O + 109H⁺

It can be seen from the above equation that:

• the construction of new bacterial cells is very inefficient: for every 55 N's used, only one is converted to new cell mass, the rest are converted to "waste" nitrite

• the process produces a lot of acid: 55 molecules of ammonium produce 109 H⁺ protons

• the process requires a lot of oxygen: every gram of ammonia oxidised requires over 3 grams oxygen

• the bacteria can grow without an organic carbon source: they use CO₂ as their carbon source

The conversion of nitrite to nitrate is carried out by Nitrobacter spp. as follows:

400NO₂^- + 5CO₂ + NH₄⁺ +195O₂ + 2H₂O -----> C₅H₇O₂N + 400NO₃^- + H⁺

It can be seen that

• oxygen requirement is much less than for the ammonia nitrite bioreaction

• biomass production of Nitrobacter is much more inefficient than biomass production of Nitrosomonas: 5,600 grams of Nitrite-N are required to make 113 gramsof cellular biomass

• acid production is also much less

• inorganic carbon is used to create cell biomass

The overall bioconversion of ammonia to nitrate is:

NH₄⁺ + 1.83 O₂ + 1.98 HCO₃^- -----> 0.021 C₅H₇O₂N +0.98 NO₃^- + 1.041H₂O +1.88H₂CO₃

This equation shows that for every gram NH₄⁺-N oxidized to NO₃^--N, 4.18 g oxygen and 7.14 g alkalinity (as CaCO₃) are used and 0.17 g of cells are produced.

As far as fish and prawn farming is concerned, nitrification is a process that can proceed in ponds to convert potentially toxic ammonia to nitrite, which can also be toxic. The process consumes TA and needs oxygen, and produces very little biomass. The nitrite in turn can be converted to nitrate. Likewise, very little biomass is produced. The issue of real importance to farmers is that the nitrogen in ammonia and nitrate is used by all sorts of microbial reactions, whereas nitrite is something of a "dead end" product, and will normally only be used if the other sources of N are depleted.

Probiotics

Probiotics is a term used to describe the application of live bacteria in order to achieve a beneficial goal. In aquaculture, probiotics have been used for some years to help improve pond water condition. The main way they work is to speed up the hydrolysis of large sized molecules and so cut down of accumulation of sludge. There is also a significant amount of evidence available that supports the idea that application of some types of bacteria can help control diseases.

The keys to getting a good result when using probiotic bacteria are

• to ensure that enough numbers of viable bacteria are added to the water, and

• to ensure that conditions are right in the pond for the added bacteria to grow

The only economic way to get enough numbers is to grow the bacteria on site from seed stock. This can be done in tanks or using bran plus some essential nutrients. The bran method is probably superior as it allows the bacteria to obtain oxygen from the air, whereas in liquid growth of bacteria, oxygen quickly becomes the limiting factor.

As far as the condition in the ponds are concerned, there must be adequate oxygenation, and there ought to be an organic source of food for the bacteria.