KNOW- HOW OF BIOFLOC TECHNOLOGY PART-2
Compiled by-Dr.M.Menaga ,Mr.M.Mohammed Faizullah Dr.S.Athithan ,Dr.S.Balasundari
For example, increased primary productivity might, in turn, lead to increased bacterial productivity in the water. This is because algae can release organic carbon that ranges from simple sugars to complex polysaccharides that can then be utilized by
heterotrophs. Moreover, algae are organisms with short lifespans, and when they die, this increases the amount of available organic carbon for faster reproduction of heterotrophs (Hargreaves 2006). However, bacteria can degrade organic materials and produce nutrients as well vitamins and other bioactive compounds that can
stimulate phytoplankton growth (Hargreaves 2006; De Schryver et al. 2008).
An inhibitory effect between microalgae and bacteria can be from the production of antagonistic growth substances such as antibiotics and/or allelopathic substances including aponin, anatoxin, microcystin and hemagglutenin (Fuentes et al.
2016). Each group can also influence the chemical environment of the other and thereby affect their metabolism or nutrient activity (Hargreaves 2006). For instance, the production of glucossidases, chitinases, cellulases and other enzymes by bacteria
might lyse the cells of microalgae (Hancock et al. 2010; Wang et al. 2010).
Phytoplankton-Nitrifying Bacteria Competitive role in Summer & Winter
There are also possibilities of substrate competition, such as for ammonia or nitrate, that depends on temperature and amount of ammonia. Phytoplankton will generally outcompete nitrifying bacteria for low ammonia-N concentrations during the summer while a higher concentration of substrate during winter will be more favourably utilized by nitrifying bacteria (Hargreaves 1997).
Heterotrophic dominance in Biofloc- Boon or Bane ?
Heterotrophic bacteria are generally classified into floc-forming bacteria and filamentous bacteria. Floc-forming bacteria encourage the aggregation of microorganisms and dead particles in the water and make the biofloc to have a small volume but high density. This might be due to production of extracellular polymeric substances (EPS), including polysaccharides that encourages binding of bacteria cells to other particles in the environment. Filamentous bacteria, however, lead to the formation of poorly aggregated bioflocs with a high volume but low density (De Schryver et al. 2008; Dauda et al. 2018a).
Density variant in Biofloc and its correlation with Biofloc colour
Biofloc density can be related to the stoichiometry analysis of Ebeling et al. (2006) who stated that 1 g of ammonia-N will produce 15.85 g of algae biomass, 0.20 g of nitrifying bacteria and 8.07 g of heterotrophic bacteria.
In addition, heterotrophic bacteria grow faster than autotrophs and the bacteria biomass produced per substrate is 40 times greater than that of chemoautotrophic bacteria (Ebeling et al.2006; Hargreaves 2006).
Biofloc colour Floc Density
| Bioflocs in algae dominated tanks are usually greenish in colour and are dominated by filamentous microalgae
(Spirogyra, Anabaena and Oscillatoria), which are usually loosely attached to each other and hence, have a spatial structure (Ju et al. 2008). |
Characterized with having a high settling volume but low density/biomass yield (Xu et
al. 2016). |
| Bioflocs composed mostly of nitrifying bacteria that are usually greenish-brown in colour | May have a lower density compared to heterotrophs dominated bioflocs. |
| Heterotrophic dominated bioflocs, will be brownish in colour and are more closely
aggregated (Xu et al. 2016). |
The highest biofloc densities are usually observed |
Why does Biofloc systems shows an increased Nitrate concentration in due course of culture?
This is because phytoplankton and heterotrophic bacteria have the ability to remove nitrogen from the system, however nitrifying bacteria only convert the toxic nitrogen metabolites to less toxic nitrate-N (Timmons et al. 2002).
Theoretically, heterotrophic dominated BFT would have better performance of nutrient conversion because phytoplankton uptake varies based on daylight exposure.
Notwithstanding, differences in nutrient removal by BFT is normally only noticed at the initial stage during biofloc establishment and thereafter nutrient removal becomes
more stable (Samocha et al. 2007; Xu & Pan 2012; Perez-Fuentes et al. 2016).
It is safe to suggest that once a matured biofloc attains equilibrium among the heterogeneous microbial community, they play complementary roles in the removal of nutrient from the culture medium. However, less toxic nitrate might still be abundant
in systems dominated by nitrifying bacteria or filamentous bacteria (Samocha et al. 2007; Crab et al. 2012). This is because filamentous bacteria store nitrate-N, which is then released under low dissolved oxygen concentrations (Su et al. 2013).
Does Heterotrophic biofloc based System alone is recommended for shrimp and Fish culture ?
Xu et al. (2016) suggested that the bioflocs dominated by a mixture of microalgae and bacteria are more beneficial than heterotrophic bacteria dominated for the culture of shrimps. As this was evident in improved growth and feed utilization performance of shrimps reared in bioflocs with a mixed proportion of microalgae and bacteria compared to heterotrophic bacteria dominated bioflocs. Ju et al. (2008) also reported a higher crude protein and lipid content of 41.9% and 2.3%, respectively, in a phytoplankton dominated biofloc-based system compared to 38.4% and 1.2% in crude protein and lipid, respectively, when bacteria dominated.
4.Influence of Bio engineering Parameter’s on biofloc characteristics
Bio engineering parameters in biofloc-based systems include temperature, mixing intensity, dissolved oxygen, pH, organic carbon source and organic loading rate which have a varying effects on the characteristics and quality of the bioflocs (Hargreaves 2006; De Schryver et al. 2008).
Temperature —- A low temperature of 4°C resulted in deflocculation of the flocs and he suggested that this might be due to the reduced activity of microbial organism lower temperatures, Wilen et al. (2003).
Krishna and Van Loosdrecht (1999) reported bulky flocs at a temperature between 30 and 35°C and suggested that this might be due to excessive activities of the microbial community in the system.
The particle aggregation and biofloc formation as dictated by the total suspended solid increased with increasing temperature, and it was significantly higher at 30 and 33°C.
A temperature of between 20 and 25°C be used to produce bioflocs with a Sludge-volume index (SVI, which is volume in millimetre occupied by 1 g of biofloc) of 200 mL g-1 (De Schryver et al. 2008).
pH and Alkalinity ——- (1) The carbon source and carbon to nitrogen ratio may have effect on the rate of decrease in pH in BFT.
➢ Dauda et al. (2017) observed a lower pH in rice
bran-based BFT compared to glycerol and
sucrose.
➢ A lower C/N ratio of 9 was reported to favour
autotrophic organisms while 18 favoured
heterotrophic organisms (Xu et al. 2016). Then,
this might suggest a rapid decrease in pH and
alkalinity in BFT with C/N ratio below 10.
(2) The choice of alkalinizing compounds may also
influence BFT and culture organisms
performance.
➢ The use of sodium bicarbonate was more
effective in maintaining alkalinity and pH in the
BFT for Oreochromis niloticus compared to
calcium carbonate, and thus further reflected in
the better growth rate and yield of the fish
reared in the system (Martins et al. 2017).
(3) Chen et al. (2006) reported that pH between
7.0 and 9.0 is required for the optimum
development of nitrifying and heterotrophic
bacteria.
➢ The use of alkalinizing compounds such as
sodium carbonate, sodium bicarbonate or
calcium hydroxide to minimize a reduction in
alkalinity to improve the buffering capacity has
been investigated (Furtado et al. 2011; Martins
et al. 2017; Zhang et al. 2017).
| BFT tanks for rearing of
L. vannamei ( Furtado et al. 2011) |
||
| Amendment | pH raise | Alkalinity raise |
| 0.06 g L-1 of
sodium carbonate, |
0.7 | 20 mg L-1 |
| 0.15 g L-1 of
calcium hydroxide and |
0.8 | 110 mg L-1 |
| 0.20 g of
sodium bicarbonate |
0.25 | 100 mg L-1 |
Mixing intensity and bio floc size–
The intensity of mixing affects the equilibrium between the rate of biofloc aggregation and breakage, thereby influencing the biofloc size
The size of the bioflocs has been found to influence their nutritional quality (Ekasari et al. 2014b)
Bioflocs >100 µm contained the highest amount of crude protein when compared with biofloc particles of 100-48 µm or <48 µm
Smaller bioflocs had the best amino acid concentrations
Compared propeller aspirator pump aerator, vertical pump aerator and diffused air blower, the diffused air blower was the most efficient in particle aggregation and biofloc formation.
Dissolved oxygen — A trend towards an increased production of more compact and larger bioflocs at higher concentrations of DO (2.0-5.0 mg L-1), this could be due to the system being dominated by floc-forming bacteria, though a much higher intensity and DO may lead to breakage of the bioflocs (Wilen and Balmer,1999)
Filamentous bacteria tended to dominate under low DO (0.5-2.0 mg L-1) conditions and this might lead to formation of less aggregated and small bioflocs but with high volume (Martins et al. 2003).
When DO becomes limiting in BFT ponds, bioflocs with
a higher SVI will be produced as a result of filamentous
bacteria dominating while a highly aerated pond with
high DO may be dominated by floc-forming bacteria and
hence a low SVI, which is better for the cultured animal.
Maintaining a high DO above 5 mg L-1 will yield bioflocs
of optimum quality and characteristics which could
improve the performance of the cultured animal
Carbon source — Ideally, the carbon source should be by-products (De Schryver et al. 2008). Most of the cheap carbon sources (rice bran, wheat, tapioca, tapioca by-product, etc.) are complex carbohydrates, but are less soluble and therefore ammonia-N removal is slower.
Potential solution to this can include fermentation of the complex carbon source (Ekasari et al. 2014a; Romano et al. 2018), which may increase the rate of utilization by bacteria or pre-treatment with other methods such as heat/or digestion with microorganisms
(Dauda et al. 2017; Romano et al. 2018).
|
C/N ratio |
It is safe to suggest that C/N ratio of 15 is sufficient for optimal performance of BFT at any point in time while C/N 10 may be a better alternative only when the bioflocs hasattained maturity. The advantage of using lower C/N ratiosbetween 10 and 15 include reduced cost of operation andsafeguarding the system from increased oxygen demandthat may lead to hypoxia condition in the culture system.
However, higher C/N ratios (>15) may be advantageous at the beginning to more quickly establish bioflocs.
C/N ratios 15 and 20 led to significantly higher disease resistance compared to C/N ratio 10 or control without glycerol addition. In this study, there were also less liver damages in C/N ratio 15 and 20 as revealed by liver histopathological examination. |
| Salinity | The growth performance of the cultured animal also showed an increasing trend with the increase in the salinity level, while the survival was significantly higher at 32 g L-1 salinity compared with the lower levels. |
Studies on the effect of different organic carbon sources on biofloc characteristics and water quality and performance of the cultured aquatic organisms
| Different carbon
sources |
Species
Cultured |
Biofloc/water
quality |
Performance | References |
| Acetate,glycerol,gluco
se and glycerol + Bacillus |
Macrobrachiu
m rosenbergii |
Glycerol and
glycerol + Bacillus produced biofloc with best nutritional quality |
Glucose and
glycerol+Bacill us led to highest survival |
Crab et al. (2010a) |
| Sugar and glycerol | None | Glycerol
produced biofloc with better nutritional quality |
Not
determined |
Ekasari et al.
(2010) |
| Cane sugar,molasses
and jaggery |
Penaeus
monodon |
Jaggery had
highest biofloc biomass and best nutritional quality |
Jaggery had
highest survival and growth |
Sakkaravarthi
and Sankar (2015) |
| Molasses,starch,wheat
flour and mixture of molassess,starch and wheat |
L.vannamei | Molasses and
starch had higher ammonia N- removal |
Molasses had
the highest growth, while mixture of all was highest in survival |
Khanj
ani et al. (2017) |
| Molassess cane
sugar,dextrose and rice bran |
L.vannamei | Molasses cane
sugar and dextrose had higher ammonia N removal |
Growth was
higher in rice bran |
Serra et al.
(2015) |
| Tapioca,wheat ,corn
and sugar bagasse |
L.rohita | Wheat had the
highest ammonia N removal |
Corn had the
highest growth, tapioca led to highest immune response and disease resistance |
Ahma d et al. (2016) |
| Molassess,tapioca,tapi
oca by product and rice bran |
L.vannamei | Tapioca and
molassess had highest ammonia N removal |
Tapioca-by-
product had highest survival and disease resistance, |
Ekasari et al.
(2014a) |
| Sugarcane
molassess,tapioca flour and wheat flour |
L.vannamei | Wheat flour led
to highest ammonia N removal,biofloc volume,chlorop hyll a,total heterotrophic bacteria and plankton concentration, as well as nutritional value |
Wheat flour
had highest growth and survival |
Rajkumar et
al. (2016) |
| Rice bran and
Molasses |
L.vannamei | Ammonia -N
removal was higher in molasses |
Rice bran led
to higher growth and survival |
Vilani et al.
(2016) |
| Glucose,starch and
glycerol |
None | Glycerol had
highest ammonia -N removal;glucose led to better nutritional quality |
Not
determined |
Wei et al.
(2016) |
| Rice flour and
molasses |
P.monodon | Rice flour had
higher biofloc volume and ammonia N removal |
Rice flour led
to higher growth, immune response and disease resistance |
Kumar et al.
(2017) |
| Molasses and
molasses + wheat |
L.vannamei | Molasses +
wheat had higher biofloc volume and ammonia N removal , but molasses had higher bioactive compounds |
Molasses had
higher bioactive compounds Molasses had higher antioxidant status, immune response and survival, but Molasses+whe at had higher growth |
Zhao et al.
(2016) |
| Glycerol, sucrose and
rice bran |
Clarias
gariepinus |
Glycerol and
sucrose led to better removal of ammonia-N |
Glycerol led to highest survival, why mass mortaities was observed in rice bran between days 16 and 18 |
Dauda et al. (2017) |
Studies on effect of different carbon to nitrogen (C/N) ratios on biofloc composition, nutrient removal and performance of the cultured aquatic organism
| C/N ratios | Species | Biofloc/water
quality |
Performance | References |
| Molasses, C/N 9, 12, 15
and 18 |
Litopenaeus
vannamei |
C/N 18 and 15
had higher biofloc biomass |
C/N 9 and 12
led to higher growth performance |
Xu et al. (2016) |
| Glucose, C/N15, 20 and
25 |
Carassius
auratus |
No difference
was observed |
C/N 20 had
highest growth performance |
Wang et al. (2015) |
| Corn starch, C/N 11, 15,
19 and 23 |
Big head carp | C/N 19 and 23
had highest ammonia-N removal |
C/N 19 and 23
had the highest growth performance |
Zhao et al. (2014) |
| Molasses, C/N 10, 12.5,
15, 17.5 and 20 |
Oreochromis
niloticus |
>C/N 15 led to
higher biofloc biomass |
C/N 10 and 15
led to higher growth performance |
Perez-Fuentes et al.
(2016) |
| Molasses, C/N 10, 15,
20, 25 and 30 |
Clarias
gariepinus |
C/N 15 had
highest ammonia-N Removal and biofloc formation |
C/N 20 led to highest growth |
Abu Bakar et al.
(2015) |
| Sucrose, C/N 15 and 20 | Litopenaeus
vannamei |
No difference
was observed |
No difference in
growth |
Xu and Pan (2012) |
| Dextrose, C/N 10, 12.5
and 15 |
Litopenaeus
vannamei |
C/N 15 led to
highest ammonia-N removal |
No
difference in growth and survival |
de Lorenzo et al.
(2016) |
| Sucrose, C/N 15 and 20 | Litopenaeus
vannamei |
No difference in
biofloc formation or ammonia- nitrogen removal, C/N 20 produced floc with better nutritional value |
No difference
in growth and survival |
Xu and Pan (2014) |
| Sucrose, C/N 15 and 20 | Litopenaeus
vannamei |
No difference
was observed |
No difference
in growth |
Xu and Pan (2013) |
| Glycerol, C/N 10, 15
and 20 |
Clarias
gariepinus |
C/N 15 and 20
led to faster removal of |
No difference
in growth and survival |
Dauda et al. (2018a) |
| ammonia-N but
C/N 10 produced bioflocs with better nutritional value |
5.Important Parameters to be monitored in Biofloc Systems
Quantitative characteristic of floc can be described as being highly porous, irregularly structured and loosely connected aggregates composed of smaller primary particles. The quantitative characteristics of floc includes Floc volume, Biological
oxygen demand (BOD), Total suspended solids (TSS), Total dissolved solids (TDS) , Total solids (TS), Volatile suspended solids (VSS), Floc volume index (FVI), Floc density index (FDI), Floc porosity, Floc size, Settling velocity and Total organic carbon.
FVI should be higher than 200 mL/g to avoid the flocs from settling too fast in regions of lower turbulence (Crab et al., (2008) and FV should be in the range of 5-50 ml/l.
Do probiotics are essential for biofloc technology?
The group of beneficial bacteria which are applied in the name of probiotics is not required for development of floc, however as mentioned earlier, the introduction of inoculum will be useful in fastening the floc development.
What should I want to do if the floc volume has exceeded its optimum level?
Incase the floc volumes are higher than the recommended level, stop adding the carbon source till it decreased. The feeding reaction can be reduced and also the supplementation of pelleted diets should also be reduced. If the level exceeds even beyond the controllable limit, draining of excess floc content is the only option.
The drained bioflocs can be later used as feed for the culture animals.
How to increase the floc volume in the biofloc culture tank?
Even after the initial fertilization for the biofloc development, if you did not see the floc, then the supplementation of carbon sources can be doubled as the premier step, followed by this, if you still didn’t see the floc, then addition of probiotic bacteria followed by the addition of minerals could balance the water for the microbes
development.
What is the optimum floc volume to be maintained for shrimp and fish culture?
For nursery culture of shrimps floc volume of around 5-7 ml/l is sufficient whereas for grow out culture 10-12ml is more than enough to run the systems under biofloc technology.
For Fish culture 10-12ml/l of floc volume for nursery culture of fishes and 25-30ml/l of floc volume for grow out culture of fishes.
How to improve alkalinity of the biofloc culture water and what is the reason behind its decrease?
As microbes use CaCo3 (Calcium carbonate) for the multiplication of cells thereby animilation of ammonia will take place without any disturbances. Due to this, a constant drop in pH and alkalinity is seen widely in biofloc systems.
This can be rectified by the addition of quick lime or any calcium forms chemicals in to the biofloc tanks. The drop in alkalinity will be highly noticed in denitrification process.
What is the easy way to maintain the floc in suspension?
The floc suspension will be always assured by supplying heavy aeration devices. This can be overcomed by maintaining the optimum floc volume. With the existing aeration adoption practices, the oxygen demand of the floc can be meet out.
Can I use feed pellets as nitrogen source for floc development?
The feed pellets of low protein diets or unused feeds can be used, however necessary care has to be taken to avoid using mold affected feeds. Besides these generally urea and ammonia sulphate can be used for improving the nitrogen level, among this two ammonia sulphate is recommended as it releases the nutrients at low level compared to urea. Also the nitrogen level is higher in urea compared to ammonium sulphate which leads to excessive nutrient discharge into the culture ponds.
Probiotics would fasten the development of floc. As biofloc itself contains mixture of heterotrophic and autotrophic bacteria the addition of probiotics is not generally recommended.In few cases where the bacterial activity may be decreased due to the
algal crash after heavy rainfall or during cloudy days the application of probiotics in biofloc systems is recommended.This will trigger the nitrification process to facilitate the ammonia removal.
Whether enzymes or aminoacids based supplementation is required for biofloc ponds?
As the biofloc contains extracellular producing bacteria the additional supplementation of enzymes are not required.The amino acid profile of the biofloc is rich in essential and non-essential aminoacids and hence external carbon supplementation would pave
way for improving the nutritional composition of biofloc.In a long run, the proliferation of microbes with a consistent carbon supply would enrich the composition of the biofloc.
How many days once we need to measure TAN &DO biofloc systems?
The biofloc systems has three transition phases in which algal based green water systems will be converted to heterotrophic based brown water systems will be converted to heterotrophic based brown water systems along with autotrophic nitrifiers. This incurs the proper management measures and hence testing of TAN and DO atleast weekly once is mandatory.As the organisation source addition is based on ammonical nitrogen, the measurement of TAN atleast three days once is recommended.
This sort of managemental measures will aid in preventing the excessive sludge accumulation in the ponds.
What is the optimal stocking density of post larvae and juveniles of shrimps in biofloc systems?
The optimum stocking density in circular tanks, raceways would be 1500 – 3000 pl/m3, whereas in nursery shrimp ponds it will be 1200 – 1500 pl/m3 for nursery shrimp culture.The recommended stocking density for grow out culture in tanks and raceways would be 300 -400 juveniles/m3; for grow-out culture in shrimp ponds will be 150 –
300 juvenile/m3.As biofloc technology involves lot of energy usage, stocking animals at higher density would be economically feasible.
What is the recommended stocking density of fish fry and fingerlings in biofloc systems?
The optimum stocking density of fish fry in raceways and circular tanks will be 80-100 fry/m3 ponds – 50-80 fry/m3 for nursery culture of fish fry’s.The recommended stocking density of fish fingerlings raceways 25-30 fingerlings/m3 whereas in lined & tanks, ponds – it would be 15 – 25 fingerlings/m3 for the grow out culture.
Ways for Sludge control
Sludge accumulation cannot be avoided, yet aeration and mixing lead to resuspension and recycling of sludge in the various biological food chain.
Sludge can be effectively drained if it accumulates on the pond bottom in a location from which one can remove it using strong water current.
You drain the sludge as long as the outgoing flow is black brown and you should stop the flow once you get clear water.
Drainage of sludge in intensive shrimp ponds is required toward the end of the cycle, when feeding is high. By that time, a weekly or biweekly drainage is recommended.
It is important to have a significant hydraulic drop from pond water level to the drainage base and wide enough tubes to drain out the sludge. Some farmers place perforated pipes on the pond bottom with the intention of collecting sludge, out to the draining canal. It seems that this method is of limited efficiency since once needs vigorous water flow to pull the sludge out of the pond bottom, a demand that is hard to achieve with perforated pipes along the pond bottom.
Pond operators are advised to periodically check oxygen at different locations and depth in the pond, to get an idea on the pond uniformity, existence of poorly aerated regions and to better know your pond.
Presence of anaerobic pockets may be critical. The response to poorly aerated sites or sites where sludge accumulate, is to re-locate aerators placement, adding aerators, targeted to sites where the existing units do not properly mix the water and the bottom. One has to be very cautious in dispersing piles of reduced sludge (black)
containing H2S.
The pH and the alkalinity should be maintained at conventional levels. Alkalinity should be above 50-100mg as Caco3/l and pH should be 7-9. Alkalinity and pH are usually stable in BFT ponds though there might be a need to add alkalinity in cases of high stocking density.
Typical floc volume are 2-40ml/l in shrimp ponds and upto 100ml/l in fish pond.
TSS (Total Suspended Solids) is one of the method to determine biofloc concentration. VSS is a conventional way of determining the organic fraction of the total solids, a common parameter in water technology.
To calculate TSS, we can use floc volume and can be calculated as, TSS = 10 X FV as a reasonable approximation.
Normal TSS values in shrimp pond water are in the range of about 50-300mg/l, while those in fish ponds reach levels of upto 1000mg/l.
When floc volume is lower than 2ml/l (shrimp) or 5ml/l (fish) in advanced stages of culture, it is advisable to add organic matters (molasses or other). However floc volume above ~15ml (Shrimp) or ~25ml (fish) may be too high. Excessive floc volume leads to increased biological oxygen demand (BOD), demanding an un-needed increase of pond aeration. Heavy load of suspended matter can lead to clogging of gills.
Settling characteristics:
Sludge can also be characterized by how well it settles. Most settling tests are conducted in a 1L-graduated cylinder. A quick and simple test to measure the sludge settle ability is developed by Mohlman (1934) and called the Sludge Volume Index (SVI). SVI is conducted with a homogeneous sludge mixture. The sludge is settled out
in 1L Imhoff Cone for 30 minutes. Settled sludge volume (V) at the bottom of the cone is measured.
Knowing the concentration of solids in the sludge suspension (MLSS) in terms of mg/L, SVI is calculated as:
Typically SVI is used without a unit, but its unit is mL/g. Typical SVI values and their meanings :
Sludge Volume Index (SVI) values: pin floc potential less than 50 ml/g
Sludge Volume Index (SVI) values: good range 50 to 100 ml/g
Sludge Volume Index (SVI) values: Filament growth 100 to 150 ml/g
Sludge Volume Index (SVI) values: Bulking at high flows 150 to 200 ml/g
Sludge Volume Index (SVI) values: Bulking 200 to 300 ml/g
Sludge Volume Index (SVI) values: Severe bulking higher than 300
