Download Powdered versus liquid bacterial preparations

Powdered versus liquid bacterial preparations
Authors: Jan Stienstra, Rob Heyboer, Siep Nagengast. Ref: V2007/20
Bacteria as "employees" of the Koi enthusiast
Bacteria play a crucial role in any koi pond in maintaining the quality of
the pond water and fish health. Bacteria are responsible for
degradation of harmful substances including ammonia secreted by the fish
and the pollution which is caused by feeding koi, plant material and feces.
In the literature this natural process always starts with the (chemo) heterotrophic bacteria and
Then followed by the autotrophic bacteria . Between those two or more groups of
bacteria an interplay is created revered to as symbiose.
The biological steps that we will deal with are known. The first step of nitrification
For example, formed by Nitrosomonas / Nitrocococus bacteria that convert ammonium
and then excrete nitrite . The second step is the Nitrobacter / Nitrospira bacteria in turn converting
this nitrite and excreting nitrate.
In a simplified form, this is as follows:
The first step
>
Nitrosomonas etc. >
NH 4 (ammonium) >
second stage
Nitrobacter etc.
NO2 (nitrite)
> finished.
> end product nitrate.
> NO3 (Nitrate)
That is the general formula for the nitrification process. Nitrosomonas Is obligate chemoautotrophic and in need of oxygen while Nitrobacter is chemolithoautotroof.
After nitrification may follow the denitrification process by which the nitrate again is
formed back to nitrite which is then converted to nitrogen gas. Nitrate is a
oxidant and an unstable component. The competition in the bacteria relevant for denitrification
is huge!
We get the following schedule for the whole process:
First step nitrification >
Second step nitrification
> denitrification
Nitrosomonas etc.
>
Nitrobacter etc.
> Denitrifying bacteria
NH4 + (ammonium) > NO2 (nitrite) > NO3 (nitrate) > NO2 (nitrite) > N2 (nitrogen)
There is a long long list of denitrifiers involved: the heterotrophic Pseudomonas sp.p,
Aerosomonas sp.p, Mycobacteria, and the bacteria Vibrio flavobacteria. In those groups
heterotrophic bacteria are the most disease-causing strains. The key question
is: what are the bacteria that control the denitrification process.
So we are dealing with these heterotrophic bacteria living in a hypoxic
environment. Such bacteria in general are found in the Bio film layer at various
types of support material and in the bottom deep layers of mud in the natural ponds.
However, as it was described at the beginning, we are also dealing with a different heterotrophic
family, These are Chemo-heterotrophic bacteria. Their role is shown in the next diagram:
Chemo-Heterotrophic > Nitrosomonas etc.> Nitrobactor etc.> [nitrate (denitrifying (heterotrophic)
bacteria)> NO2 > N (gas)]
All biological processes in the water start with Chemo-heterotrophic bacteria, only
later the autotrophic bacteria play a role and much later the heterotrophic
denitrifying bacteria. When starting a pond you should consider them
account. The Chemo-heterotrophic bacteria need oxygen, being as it were
oxygen Slurpers. Here lies one of the differences between Chemo-heterotrophic bacteria who
need of oxygen (areoob) and the heterotrophic bacteria involved in the
denitrification process. The latter are loving oxygen (anaerobically).So We are therefore
Talking of two different types of groups that are identified as heterotrophic
bacteria. We should not exclude the chemo-heterotrophic bacteria out of aquatic life.
They are native to the aquatic life, and belong there both in nature and in the pond.
Nitrifying bacteria.
nitrifying bacteria are Autotrophic. It is not possible to trade autotrophic bacteria in
Powdered form. That's why nitrificende bacteria are always soled as a liquid
product. Nitrifying bacteria can survive a certain time without oxygen. The
lower the ambient temperature, the longer the bacteria can survive without oxygen. In
an average temperature of 15 ° C the life of the bacteria in a closed bottle is about 5 to 7 months.
In the past, people often assumed that these classical nitrifying bacteria where the only bacteria that
determined the quality of pond water . Today we have to note and except however, the fact that
more than 3600 bacteria are involved in the present nature who all in one way or another participate
in the nitrogen cycle. Many of these bacteria have not yet been named. The investigation of these
unknown bacteria will require more research for years to come.
Chemo-heterotrophic bacteria.
The chemo-heterotrophic bacteria are due to their unique properties used in various industrial
applied organic 'production' processes. This Article has focused mainly on the role of chemoheterotrophic bacteria in the very first biological steps in the pond.
Chemo-Heterotrophic are spore-former. The spores are resistant to dehydration, can therefore be
traded in powdered form. The spores spread through the air, ground (eg via shoe soles), and through
water. These bacteria get their energy from organic material, that is their food source.
Explanation of terms
Perhaps it is good to explain some terms used, what do we mean with Chemo-heterotrophic and
heterotrophic, and what is organic?
an organism is Heterotrophic if it builds it's organic cell material from organic
substances made by other organisms. The heterotrophic organism therefore depends on
other organisms,it disimilates molecules who were assimilated by autotrophic organisms. This
includes a wide range of organisms, the simplest heterotrophs are yeasts. But
also the human is heterotrophic to be precise chemo-organoheterotroof. The word "heterotrophic" is
derived from Greek and literally means "nourishing themselves from others" (heteros "strange ", "another" hit - "food"). Heterotrophic is the opposite of
autotrophic. Most plants are autotrophic and can use sunlight, carbon dioxide
from the air and minerals to grow.
A Chemoheterotroof organism is an organism that obtains energy through biological
redox reactions between inorganic substances. Often in life processes, energy in form of
redox reactions is stored and transformed. The energy generation (the
catabolism) is usually a redox reaction. This energy is used for growth. If the
same substance is partially oxidized and partially reduced it is called
fermentation. A Chemotrophic organism is an autotrophic or heterotrophic organism that obtains the
energy required by oxidation of chemical compounds known as chemosynthesizing. These
compounds can be both organic and inorganic.
The name giving comes from Greek. Chemo means "chemical" and troph means "food ".
Auto means "self" and hetero represents "other". Chemotrophic organisms who live completely or
partialy of organic compounds are called chemo-heterotrophic, chemo-organotrophic or
organotrophic and those living of inorganic compounds chemoautotrophic, chemolithotrophic,
lithotrophic or chemo-litho-autotrophic.
Organic is the term used for compounds known to contain both carbon atoms as
hydrogen atoms and are found in living beings. The opposite are the
inorganic or mineral compounds such as ammonium, nitrite and nitrate.
Carbon, the basis of all organic compounds, is just one of the 92 natural
elements, but the compounds of carbon (typically with hydrogen, oxygen, nitrogen,
sulfur, etc.) are still more numerous than those of all other elements.
Chemoautotrophic (or chemotrophic autotrophic) organisms, who obtain their energy by chemical
reactions from inorganic compounds, make all necessary compounds from carbon dioxide. Most are
bacteria or archaea, who can live in extreme conditions. Scientists believe that these organisms are
the first who colonized earth and as a byproduct delivered oxygen. Baccilus subtilis is an example
of a bacterium that can live under extreme conditions. Bacillus subtilis is nonpathogenic and is the most famous chemo-heterotrophic spore vorming, gram-positive bacteria.
These bacteria are aerobic (oxygen-requiring bacteria) occurring in the soil, water, air, plants,
animals and even humans. Baccilus subtilis is also the largest identified bacteria strain in the world.
It is the most studied and therefore stricken, bacteria on Earth. Even a trip around the planet Mars
was part of the research. These bacteria are mainly extracted from the ground and are produced by
bio-industrial processing in large quantities. It is used for the pharmaceutical, agricultural and food
industries, as sludge remover for water treatment plants, and ... .. it is also used for koi ponds. Japan
and America are the largest producers of Baccilus subtilis sp.p. tribes. There are 18 different
Baccilus subtilis sp.p. strains, of which 3 strains are spore-forming.
Literature study shows Bacillus subtilis sp.p particular characteristics. It is a workhorse which can
excrete more than 7 types of bases (proteins or enzymes) , and even more types of saccharides.
Polysaccharides are carbohydrates made up of many monosaccharide units. Lipo-saccharides
consist mainly of fats and oils. The excreted substances are used in the pharmaceutical industry
to produce medicinal products (eg vaccines, detergents, insulin, antibiotics, probiotic
resources, and medication)
next to that there stil is research performed today with elucidating the special shaped proteins
(enzymes, SPase) that these bacteria can produce.
"In many organisms there is but one Spase present. Harold Tjalsma discovered that B. Subtilis
poseses no less than seven SPase. Important finding is that these enzymes with a
seemingly identical functions are involved in various cellular processes. Three SPase
proved essential for the viability of B. subtilis cells. Surprisingly one of the non-essential SPase
showed a large similarity with enzymes occuring in higher organisms including humans. This is
why in the future this type of Spase in a relatively simple bacterial model system can be researched
further
Source: RUG – Groningen.”
Bacillus subtilis, a very strong bacteria.
Bacillus subtilis is resistant against (radioactive) radiation and UV radiation, high temperature
(up to 120 ˚ C) and low temperature (4 ˚ F) and is immune to certain chemicals such as
salt and PP treatment!. They can also endure large pH fluctuations. In addition,
these bacteria can also be used in the processing of various metallic
contaminants. His brother the Bacillus licheniformis takes care of nitrate reduction.
Bacillus subtilis also has another more sinister feature. It is a
cannibal, a lack of specific nutrients will make the Bacillus subtilis spray of
off antibiotics, then eats his own family, and goes to sleep.
Green bacillus subtilis are viable bacteria.
Red bacillus subtilis bacteria are lifeless. Green
eat the red bacteria, and then go to sleep.
Source: José E. González- Pastor, Unidad de Genética Molecular, Hospital Ramón y Cajal,
28034 Madrid, Spain.
What does bacillus subtilis do with ammonium in our pond.
Bacillus subtilis is ubiquitar (common in nature) as a nitrogen binder it draws
nitrogen (N) from the air, water, soil and plants.
Bacillus subtilis does not break down ammonia, but it binds it to one or more of the 7
enzymes secreted by a complex biochemical conversion. All these
biochemical biochemical reactions give off substances which are needed in the growth of such
plants. Ammonium in these biochemical reactions very rapidly degraded.
Accompanying graph show the decrease in ammonia at different temperatures.
When starting a pond, these Baccilis subtilis sp.p is applied. At the start
There are no autotrophic bacteria. This is because after a short time, there's no more nitrogen
nutrients available for the development of autotrophic bacteria. There are
also biochemical means in this case as a nutrient sufficient to autotrophic bacteria to develop. The
nitrogen-containing compounds such as NO2
-N (nitrite-nitrogen),
are oxidized through a reductor to the higher nitrate compound. A reductor is a chemical compound
who can donate electrons in a chemical reaction. The substance who absorbes the electrons is
called oxidant. Not only the nitrogen dissolved in water (air that is bubbled through the pond
contains 20% oxygen and 80% nitrogen), but also carbon and nitrogen present as glucose and
phosphate in organic material is a source of nutrients for the Bacillus subtilis to develop.Organic
material can be composed of plant debris, inactivated bacteria, excess koi food remnants and faeces.
But also from free-floating living microorganisms.
We distinguish two types of bacteria present in koi ponds.
- Free-floating bacteria
- Bacteria that are trapped in biofilms.
Free-floating bacteria are bacteria "swimming around" free in the water. When we
take water samples, the samples will contain free floating bacteria. Also the
nitrifying bacteria can be present free floating in the water. There have been many
investigations into the Bacillus subtilis in relation to the nitrifying bacteria.
Indeed, it is assumed that chemo-heterotrophic bacteria enter the competition with the autotrophic
bacteria and supersede them. Here it is shown how the Bacillus subtilis supersedes this
free-floating nitrifying bacteria.
In the now "deceased" Dutch Koi company Amikoi the water in a basin with a large
fish population was studied. The water of this basin was filtered over Japanese mats.
DBI test shows after 4 days.
a Heterotrophic activity
DBI test shows the first step
nitrifying activity
Nitrite detection
Hanna Instrument
This DBI test shows an example of the first step nitrification (conversion of ammonium
to nitrite). On the left picture it shows the heterotrophic activity after 4 days. That this took 4
days indicates that few heterotrophic bacteria were present and that the
autotrophic bacteria are dominant. The heterotrophic test can not determine which
heterotrophic bacteria are concerned. Practice has shown that the baccilis subtilis sp.p
rapidly converts ammonium. Below are pictures of a similar test
performed after an incubation period of 5 days in the same pond water, but after adding
of baccilis subtilis bacteria and aeration.
Heterotrophic activity after 3 hr No first step nitrifying
turnaround
bacteria present
No nitrite
The results show that the chemo-heterotrophic bacteria have repressed the nitrifying bacteria. Yet
the nitrite disappeared, which was confirmed by
Hanna water analysis measurements on both water solutions.
The above results leads to the following schedule for the nitrification of
pond water:
For free-floating bacteria:
Chemo-Heterotrophic > Nitrosomonas > Nitrobactor > [nitrate (denitrifying
(heterotrophic) bacteria) > NO2 > N (gas)].
By adding Baccilis subtilis the first step nitrifying bacteria in free
floating form is no longer available.
What possibly remains is now:
Chemo-Heterotrophic > Nitrobactor ed > [nitrate (denitrifying (heterotrophic) bacteria) >
NO2 > N (gas)].
You may ask yourself, nitrifying bacteria are still important for our ponds because
they nevertheless provide the first step of nitrification. That's true but they are mainly
in bioflms and not free floating form. Think about it in your own practice situation.
When your pond water passes through the UV tube, or if you apply an ozone plant.
Do the free-floating bacteria Survive? Baccilis subtilis will survive it.
But the first step nitrifying bacteria then? Is that maybe the reason that nitrite is found in the water
when free floating Nitrosomonas are present? And does this means that the second step of the
nitrification which converts nitrite to nitrate is missing? These are the questions the preceding can
evoke.
Therefore further investigation was needed. This research was made possible by the
faculty of Micro Biology at Radboud University Nijmegen. In a subsequent article
on the second stage nitrification research, moving bed material K1 and K3 was further
investigated. the product Biogro bacteria was used as a startup application. Patrick Scheepers has
already written that "the biofilm is the" brown gold "of the koikeeper." the following article is
therefore dedicated to Patrick Scheepers.
Bacteria in biofilm.
Recording the first and the second step nitrifying bacteria in a biofilm can only be done with
biochemical analysis in combination with FISH microscopy, Electron Microscopy or other complex
systems analysis. It is generally assumed that almost all the nitrifying bacteria feel at home in a
biofilm. The chemo-heterotrophic Baccilis subtilis (BS) bacteria were added to the water freefloating where they surpprese and supersede the free floating nitrifying bacteria but not the
nitrifying bacteria present in the biofilm
More on this later.
For now the processes in the biofilm are presented schematically as:
Chemo-Heterotrophic > Nitrosomonas > Nitrobactor > [nitrate (denitrifying
(heterotrophic) bacteria) > NO2 > N (gas)].
As said before more on this later.
Saccharides.
The baccilis subtilis secreted substances in the form of polysaccharides and / or liposaccharides. Or the combination of both. Polysaccharides are insoluble in water.
This jelly-like substances are found in the biofilm. The pictures demonstrates the
formation of these substances in the product: Kamiac of House of Kata.
A Small drop of bacteria Kamiac B
solution is placed on a culture medium
Within 2 hr a jelly-like
deposits is formed.
We also find these products In filter materials, In siporax filter medium (Photo: left) the slippery
layer is clearly visible and also in Kaldnes K1 (pictured right) there is a clear colorless deposit to be
seen. With the naked eye, these colorless gelatinous layers are difficult to see. On both filter media
it required up to 3 weeks for this gelatinous layer to develop into biofilms.
Kamiac powdery substance.
House of Kata takes a powder "bacteria " with nutrient on to the market.
We do not exclude that there are multiple brands, Who contain these powder
"bacteria". we have put the word Bacteria, in quotation marks because there were doubts about that
product. Those doubts were taken away after thorough research and study found that it is a sporeforming Baccilis subtilis strains. How does that work and do we see that too? This Requires the
Use of a Scanning Electron Microscope (SEM) whith which the stages of the spore forming bacteria
from "birth" to the "new growth" could be captured.
Furthermore, a culture test was performed on specific culture media.
During the SEM study, diatoms were found (see images below).
Meaning we are dealing with a purely natural product. Diatoms are found in the soil and/or the
aquatic environment
The picture below shows an overview of the Kamiac product.
Kamiac
Besides the cyst, a closed "shell-like" envelope (yellow arrows) the powder sample contained
calcium-rich mineral (the tiny grit shown by the red arrow).
Kamiac product
an enlargement of a cyst with in the background and below,calcium-rich minerals (calcium)is
shown Above. This cyst is about 0.1 mm long.
Below is an image of a culture test used to looks at the emergence of "bacteria" from such a cyst.
Kamiac
An isolated cyst that was incubated in a nutrient medium. The cyst swells from 0.1
mm to ± 0.5 mm and cracks open at the bottom. The spore forming bacteria
are released to the culture medium (see red ring around the cyst note).
Kamiac
Finally the cyst disappears, leaving a colony of the BS sp.p. tribes behind.
Kamiac
SEM image of the ruptured cyst of which the spores are already 'released'. This
recording is 200 x enlarged.
Kamiac
Magnification of a spore from the cyst (magnification 60.000x!)
literature information:
Construction of a Bacillus spore Subtillis. Photo right the baccilis subtilis that made a trip
to the planet Mars. (Source: Charles P. Moran, Jr.., Emory University School of Medicine.)
The spore consists of an inner wall, called the cortex or bark, a very thick layer of a special kind of
mucosal peptide. This layer resists the very high pressure state on the cell membrane. This high
pressure is caused by the low water content in the sporeprotoplast. By Diffusion the water
molecules want to go in which makes the sporeprotoplast tends to swell, this "expansive force"
needs to be stopped by the cortex. The low water content is partly responsible for the
heat resistance. Proteins are difficult to break without water. In addition, the proteins in the
protoplasm are constructed"stronger" so they are more heat resistant than a normal cell.
The outer layer is the spore coat which exists out of a keratine like protein. This coat is
responsible for resistance to chemicals such as disinfectants, but also from exo-enzymes from other
bacteria in the environment (which prevents the spore from prematurely
decay). When the spore coat is formed the cell obtains his light refracting properties. The
heat resistance develops only after the protoplasm has formed large amounts of dipicolinic acid (a
substance which does not occur in normal cells)and a lot of calcium ions were absorbed
Kamiac
The yellow arrows indicate the spore formers. The red arrows indicate the (rod-like) vegetation.
The green arrow points to secreted enzymes. "Activation of the spore-former
happens if the environment becomes more favorable for the bacteria. After the activation follows
the germination and outgrowth (reproduction, vegetation). The activation probably occurs by
heating. Calcium, dipicolinic acid and mucosal peptide fragments are excreted and the bark is
aborted. "Source: Wikipedia.
Some gram-positive bacteria in response to starvation form spores . The
spore forming process takes 10 hours. A shortage of Guaninenucleotiden in the cytoplasm of the
vegetative bacteria creates a dividing of the protoplasm followed by an endocytosis-like process,
forming the spore. The DNA is replicated
and the membrane or sporeseptum by invagination of the mother cell membrane between a DNA
molecule and the rest of the cell with the other DNA molecule begins to form.
The plasma membrane of the mother cell surrounds the sporeseptum and forms a
double membrane around the DNA. The now formed structure is called a pre spore.
During the above process calciumdipocolinaat is formed in the pre spore . Finally
the bark is formed and the rest of the vegetative bacterium degraded. The
spore-former contains only 10 to 30% water compared to a bacterial cell.
Kamiac
Sporeseptum starts with recess (yellow arrow)
Kamiac
The green arrows are the spore-former. The yellow arrow indicates a secreted enzyme. The red
arrow are vegetative bacteria, say youngsters. The blue arrow points to a more grown vegetative
bacteria.
Kamiac
The red arrow points to the vegetative bacteria. The yellow arrow indicates the secreted enzymes. In
the green arrows, new spore forms by invagination from the vegetative bacterium. This cycle is
repeated continuously when sufficient nutrients are present.
Kamiac
The red arrows point to the bacteria in the vegetative state. The yellow arrows point to
The secreted enzymes produced by the bacteria .
Kamiac
V = 24000 X
With enough nutrients available the vegetative bacteria develop to a total outgrowth. The SEM
image above shows the overall growth of the baccilis subtilis sp.p. bacteria strains.
Kamiac product V=50,000 X.
Detail of the rod-like bodies Baccilis subtilis sp.p.
Kamiac product
The above photo shows the mature colony baccilis subtilis bacteria developed
on a specific agar (culture medium) for baccilis subtilis bacteria.
Algae reduction by baccilis subtilis bacteria.
As Known the various Baccilis subtilis excrees enzymes. These enzymes can lead to incineration
and / or other biochemical reactions. It shows that this baccilis subtilis can contribute in reducing
algae growth.
The images below was made with confocal laser scanning microscopy.
The orange / brown colored pearls are
algae. The cysyt are blue / green.
Recorded 2 hours later, the amount of algae
is reduced. The blue / green colored
cysts contain cavities, the spores are therefore
released and begin to activate.
The effect of FP treatment on the bacterial life:
The Koi hobbyists who according to an expert diagnosis, should apply a Potassium
Permanganate (PP) treatment can actually see for themselves how Bacillis Subtillis sp.p.
bacteria reactivate in their pond. In the present study the product
Kamiac of House Kata is used after PP treatment. 6 Hours after PP use the treatment creates a
brown color in the water sometimes followed with oxidized organic material floating at the surface.
Then, if necessary, after about 6hours an additional booster doses is used to finish the cure.
Normally, the PP treatment ends after 12 hr.
Then hydrogen peroxide is commonly used to decolour the pond water. If you are not using
hydrogen peroxide, it takes between 1.5 up to 2 weeks before the water is somewhat clearer. But
when for example Instead of hydrogen peroxide you apply the Kamiac bacteria product according
to the House of Kata instructions you will see crystal clear water starting to apear within 12 hours
Bacillis Subtillis strain (s) amongst other substances excreed peroxidase. Peroxidase (POD) is an
enzyme (not to be confused with the chemical compound of hydrogen peroxide). Hydrolase, lyase,
Isomerase, Ligase, Amylase, transferases and Oxidorerductase, etc. (with enzymestudy you
frequently encounter the output - ase-) are enzymes involved in biochemical
reactions. Peroxidase enzyme acts as a catalyst in the formation of free radicals
(eg an oxygen radical that can act as oxidant). The bacteria do need some time to multiply to
achieve a sufficiently large population (by a deviding process). But 12 hours after PP treatment the
same effect is achieved as in the case that hydrogen peroxide was added. The aforementioned free
radicals also transform nitrite by oxidation to the higher nitrate compound.
Chart nitrate reduction at different temperatures.
The enzyme peroxidase is found in:
- higher plants
- Pineapple
- Potatoes
- Pulses
- Cereals
- Sugar beet
- Figs
- and bacteria, such as the aforementioned Bacillis Subtillis bacteria.
In a Kamiac addition, the UVC and Ozone in principle, may persist. This is because normally the
dose of ozone and the UV radiation dose are low. The free-floating Bacillis Subtillis spore forming
and the mature bacteria in the water column can normally withstand the ozone and UV. The
released enzymes will however be oxidised mitigating the effect of peroxidase. Usually, therefore it
is advised that the ozone, and UVC are turned of for 48 hours so the enzymes remain intact. If you
own an ozone installation with an integrated redoxmeter you can temporarely turn the ozone of and
still read the redox value.in an average pond where UV is used the redox value is normally between
130-160mV after the kamiac bacteria are added the redox value wil first drop to About 120140mV , after about 12 hours it will start tp rise to a redox value of 230-280mV provided the UVC
and ozone are turned of
Baccilis subtilis produce enzymes that have a probiotic effect. a specic strain can be isolated from
multiple families and their enzymes who can subsequently be stored in a liquid solution. specific
biochemical solutions are used who do not affect the enzymes and can extend the viability of the
enzymes / bacteria. This is only possible if they are spore forming bacteria and these products are
only successful when applied in ponds where several nutrient media are present.
In a subsequent article, "The biofilm is the" brown gold "of the koikeeper" the
results of a 2-year research which was conducted at Koipaleis Arnhem are presented. Here the
Nexus moving bed system is investigated. The Nexus systems were started with Bacta totaal
in the first year and at a later moment Biogro was used
In this study it was particularly crucial to know if there is an overall degradation process within the
biofilm on the suportmaterial Kaldnes type K1 and K3.
Arnhem 25th September 2007.
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