How to grow your own denitrifying bacteria, the method so far...

Discussion in 'Reef Aquarium Articles and How To's' started by invert phil, Jan 30, 2010.

  1. invert phil

    invert phil Millepora

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    How to grow your own denitrifying organic bacteria

    I have developed a method using lawn fertiliser which is the method I am currently using and am about to try a second method which should be more controllable. This second method will require potassium phosphate and sodium nitrate.
    I originally started my bacteria culture using tap water and a weak house plant fertiliser but the nitrate, phosphate and potassium (NPK) levels were too low and resulted in a very weak amount of bacteria. I have recently been using lawn fertiliser which is much more concentrated in NPK with great results. The NPK ratio of the lawn fertiliser is 38-5-5.

    Materials:
    2 Glass jars ca.200mL
    Small plastic syringe
    Lawn fertiliser with N-P-K ratio around; 38-5-5
    Sugar
    Synthetic seawater using RO water at ca.33ppt
    5mL mature tank water
    Glass 500mL jug
    PO4 and NO3 test kits
    Spoon scoops from supermarket (need ½ teaspoon scoop)

    Method:
    Add 300mL of sea water to one of the glass jars using the glass jug. Then add a level ½ teaspoon scoop of lawn fertiliser, put lid on the jar and swirl till dissolved (the water may have a green tinge), this will be our stock solution. Aerate for ca. 1 hour to oxidize any free iron which could cause problems later. Now fill the other jar with 250mL of seawater and add 50mL of the stock (seawater from the first jar). This new 50mg/L as NO3 seawater media will be your bacteria media. Now take a sample and measure NO3. If you are unsure whether it is 50mg/L you can double check by taking 20mL of bacteria media and adding it to 80mL of seawater and testing it for NO3, this sample should contain 10mg/L NO3. If it doesn’t, you can adjust the bacterial media by either diluting it with sea water or adding more stock fertiliser till at around 50mg/L as NO3. Now add two table spoons of sugar to the bacteria media and swirl for about 30 seconds till some sugar is dissolved (there will be a sugar layer at the bottom; this is okay). Now add ca.5mL of mature tank water using a syringe to the bacteria media and close the lid securely. Swirl the jar daily and Monitor NO3 and PO4 content every 2 days until at 0mg/L this can take one to two weeks. It is very important that the jar is stored in the dark at room temperature to allow to ferment.
    When at 0mg/L you have grown your first culture of bacteria. Now you need to repeat the procedure, adding 50mL of the stock from before to 250mL of seawater in a jar but instead of adding a mass of sugar only add a teaspoon. Inoculate the new media by adding ca.5-10mL of the bacterial media from before, and then leave in the dark at room temperature again. Monitor NO3 and PO4 after 24 hours. If NO3 concentration and PO4 isn’t at 0mg/L add another teaspoon of sugar and leave for another 24 hours, repeat NO3 and PO4 test and one teaspoon of sugar addition every 24 hours until PO4 and NO3 are at 0mg/L. It should only take 2-4 days to reduce down to 0mg/L.
    Once at 0mg/L this bacterial solution is ready to dose into your tank at a rate of around 3-6mL per 100 Gallons with carbon source daily until cyanobacteria is gone or just as a bacterial top up once per week. The bacteria should be stored in the fridge for up to 3 months, chilling the bacteria should put them into a dormant state but not kill them.
    Phosphate in lawn fertiliser is in the form phosphorus pentoxide (P205) it cannot be measured by a phosphate kit (PO4) yet, when bacteria have broken down the majority of the nitrate they will break down phosphorus pentoxide to phosphate, this has been observed in my PO4 testing when the nitrate level in the bacterial media has been reduced to around 2.5mg/L - 5mg/L with a 1mg/L PO4 reading.

    Organic bacteria will use oxygen whilst feeding on the sucrose until the oxygen level is completely depleted then use nitrate as a form of oxygen to respire while feeding on the sucrose. The first fermenter is used to get a good volume of bacteria and then the second fermenter is inoculated from this bacteria and carbon source slowly added to try to avoid too much excess sugar in the solution so that it can be stored.

    <o:p> </o:p>
    Why grow and dose your own cultured bacteria?<o:p></o:p>
    Growing bacteria yourself instead of buying it not only saves you money, it has the added benefit of enhancing the organic bacteria already in your tank. This means that you’re not adding foreign bacteria that probably won’t survive in the conditions of your tank. It has been observed that bacteria can feed on other bacteria so even if there are dead bacteria they will be fed upon and aid your bacteria in your tank. Any excess dead bacteria not eaten by other bacteria will feed sponges and other filter feeders in the aquarium. <o:p></o:p>
    <o:p> </o:p>
    These heterotrophic organic denitrifying bacteria require an organic carbon source to grow therefore this type of bacteria is best used when dosing an organic carbon source like vodka, vinegar or sugar. It is near impossible to know how much organic carbon to dose for each individual tank, but by adding small amounts of organic carbon and increasing this over time and monitoring nutrients it is possible to work out how much is required. Also to stop cyanobacteria from competing for the nutrients it is possible to stop their numbers from building up in the first place by dosing bacteria to outcompete them when it comes to nutrients<o:p></o:p>
    <o:p> </o:p>
    The carbon source and bacteria is best dosed in front of a pump connected to a reactor such as a zeolite, GFO, GAC or fluidised reactor as these types of media within the reactor provide the ideal habitat for aerobic and anaerobic organic bacteria. Ideally these reactors should be used in the dark to avoid any competitive algae benefiting from the organic carbon dosing. I currently dose bacteria and a mix of vodka vinegar and sugar into my zeolite and GAC reactor.<o:p></o:p>
    <o:p> </o:p>
    How safe is the fertiliser?<o:p></o:p>
    I have found lawn fertiliser to be the ideal nitrogen to potassium to phosphorus ratio. It is high in nitrogen which is ideal for bacteria to quickly reduce. Fertiliser sold in the UK has to have a statutory fertiliser declaration this has to state what is in the product. The only heavy metal stated on the box of the lawn fertiliser I use is; soluble iron chelated by EDTA. I am not sure what form the iron is in therefore I have aerated for one hour just in case. This will oxidise any free iron making it safe (just in case there is no or little iron bacteria in the tank to utilise this although I suspect that there will be some). As I mentioned before, I run zeolites and activated carbon in my tank, so any metals should get absorbed. Just to be clear the amount of fertiliser dosed is extremely diluted and not to mention bacteria treated. <o:p></o:p>
    Has this method been thoroughly tested?<o:p></o:p>
    No, this is a method that I have developed with fellow freshwater and marine microbiologists. It is something that I use in my own tank with a high fish bioload and softies. I have not used this bacterial fermenter and dosing of the resultant bacteria with any hard corals. As mentioned in the method I have used a weaker fertiliser originally but didn’t feel this was strong enough in terms of bacteria biomass. I have only been using this stronger version of the bacteria for around a month and have been dosing at a rate of 10mL per day (5mL in the morning and the same in the afternoon into my 87 gallon system) to see the effects of dosing in excess. It is still only early days and it is still in its trial phase.<o:p></o:p>
    <o:p> </o:p>
    What types of bacteria will the fermenter culture?<o:p></o:p>
    This organic bacterial fermenter creates an ideal environment for both aerobic and anaerobic bacteria. The media in the fermenter will start as a fairly oxygenated environment, as organic aerobic bacteria start to multiply and break down the sucrose they will use up the oxygen. This can take a while with the original fermenter, but once you have inoculated the second fermenter with bacteria from the first one this process will happen very rapidly. The beneficial heterotrophic denitrifying bacteria require an anaerobic environment plus a carbon source to reduce phosphate and nitrate during respiration. Bacteria will work together and compete with one another to break down molecules. This means that when you dose this concentrated bacteria you are dosing aerobic and anaerobic bacteria. Most of these bacteria will be in a dormant state due to being placed in the cold temperatures of the fridge. There will also be a lot of bacterial spores from these bacteria which is something else that you’ll be dosing when you use this method.
    Most heterotrophic denitrifying bacteria are in a dormant state in aerobic conditions, a small number of denitrifying bacteria in freshwater can utilise sucrose in an aerobic environment but this is yet to be proved in a marine environment. There are bacteria that use carbon aerobically but require an aerobic environment and are dormant in an anaerobic environment. This dormant phase is the same for anaerobic bacteria when they are in an aerobic environment. This has been seen in many scientific papers looking at the types of bacteria found in the open ocean. It was also found that anaerobic bacteria in the open ocean are a lot more abundant than the aerobic types.




    Last edited: Jan 30, 2010
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  3. steve wright

    steve wright Super Moderator Staff Member

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    very interesting article and research Invert Phil

    Steve
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  4. mirandacollc

    mirandacollc Flame Angel

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    can you come make my lawn grow better?
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  5. invert phil

    invert phil Millepora

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    I don't even have a lawn :(
  6. mirandacollc

    mirandacollc Flame Angel

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    mine is all kentucky bluegrass and I was wondeing with that fertiliser if you can help. I fertilise 4 times a year and stripe it like fenway yes I said fenway park. I just want it to be 100 percent. does this count haha. I am messing with you. Glad to see your making this article.
  7. invert phil

    invert phil Millepora

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    lol :) Thank you for the nice comments :)
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  9. rohitratnakaran

    rohitratnakaran Astrea Snail

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    This i will be giving a try. Nice article.