**3.2. The role of microorganisms in BFT aquaculture systems**

Microorganisms play a key role in BFT systems. The maintenance of water quality, mainly by the control of bacterial community over autotrophic microorganisms, is achieved using a high carbon-to-nitrogen (C:N) ratio, since nitrogenous by-products can be easily taken up by heterotrophic bacteria. High carbon-to-nitrogen ratio is required to guarantee optimum heterotrophic bacteria growth, using this energy for maintenance (respiration, feeding, movement, digestion, etc.) but also for growth and to produce new bacterial cells.

The stability of zero or minimal water exchange depends on the dynamic interaction among communities of bacteria, microalgae, fungi, protozoans, nematode, rotifer, etc. that will occur naturally. Such consortia of microorganism will help on the water quality maintenance and recycling wastes to produce a high-value food. In a study with stable isotopes, Burford et al. [12] estimated a daily nitrogen retention of 18–29% into the shrimp obtained from biofloc biota, while Avnimelech and Kochba [26] found about 25% of assimilation for tilapia, using the same technique.


**Table 1.** Main water quality parameters monitored in BFT systems and its ideal and/or normal observed ranges.

Organic matter and nitrogen wastes are a huge problem in aquaculture. Phytoplankton, heterotrophic, and nitrifying bacteria have the most important role in the nitrogen and OM reutilization. Fungi, ciliate, protozoa, rotifer, copepod, and nematode complement the biofloc community, participating in the recycling of organic matter as a part of complex food webs which include the cultured species.

Mutualism and commensalism relationships occur among some group of microorganisms in BFT, e.g., bacteria-bacteria or bacteria-microalgae. In low water exchange cultures, complex biofilms are generated in which coexist heterotrophic and nitrifying bacteria. Inorganic ions are attracted to the surface of these biofilms and the solid surfaces of the substrate, promoting greater nitrification processes [27]. Some bacterial strains have a positive effect on microalgae growth not only for planktonic species but also on attached (benthic) species [28]. The extracellular polysaccharides of benthic diatoms may be used by heterotrophic organisms as carbon source [29].

One current practice in BFT is the use of commercial bacteria consortia (probiotics). The main reasons of probiotics used in BFT are (i) help to stabilize the heterotrophic community and to compete with autotrophic microorganisms (mainly in the initial phases), (ii) help to recycling the organic matter, and (iii) control solids and TAN levels.

### *3.2.1. Bacteria*

Organic matter and nitrogen wastes are a huge problem in aquaculture. Phytoplankton, heterotrophic, and nitrifying bacteria have the most important role in the nitrogen and OM reutilization. Fungi, ciliate, protozoa, rotifer, copepod, and nematode complement the biofloc community, participating in the recycling of organic matter as a part of complex food webs

**Parameter Ideal and/or normal observed ranges Observations**

60% of saturation

Temperature 28–30° (ideal for tropical species) Besides fish/shrimp, low temperatures

pH 6.8–8.0 Values less than 7.0 is normal in BFT

Salinity Depends on the cultured species It is possible to generate BFT, e.g., from

TAN Less than 1 mg L−1 (ideal) Toxicity values are pH dependent Nitrite Less than 1 mg L−1 (ideal) Critical parameter (difficult to control).

Nitrate 0.5–20 mg L−1 In these ranges, generally not toxic to

Orthophosphate 0.5–20 mg L−1 In these ranges, generally not toxic to

Alkalinity More than 100 mg L−1 Higher values of alkalinity will help the

(tilapia fingerlings) and 20–50 mL L−1 (juveniles and adult tilapia)

**Table 1.** Main water quality parameters monitored in BFT systems and its ideal and/or normal observed ranges.

For correct fish, shrimp, microbiota

but could affect the nitrification process

Special attention should be done, e.g., on protein level of feed, salinity, and

nitrogen assimilation by heterotrophic bacteria and nitrification process by chemoautotrophic bacteria

High levels of SS (measured in Imhoff cones) will contribute to the DO consumption by heterotrophic community and gill occlusion

(~20° C) could affect microbial

respiration, and growth

development

0 to 50 ppt

alkalinity

the cultured animals

the cultured animals

Dissolved oxygen (DO) Above of 4.0 mg L−1 (ideal) and at least

94 Water Quality

Settling solids (SS) Ideal: 5–15 mL L−1 (shrimp), 5–20

Total suspended solids (TSS) Less than 500 mg L−1 Idem to SS

Mutualism and commensalism relationships occur among some group of microorganisms in BFT, e.g., bacteria-bacteria or bacteria-microalgae. In low water exchange cultures, complex biofilms are generated in which coexist heterotrophic and nitrifying bacteria. Inorganic ions are attracted to the surface of these biofilms and the solid surfaces of the substrate, promoting greater nitrification processes [27]. Some bacterial strains have a positive effect on microalgae growth not only for planktonic species but also on attached (benthic) species [28]. The extracellular polysaccharides of benthic diatoms may be used by heterotrophic organisms as carbon source [29].

which include the cultured species.

The heterotrophic bacteria use the organic compounds as a carbon source. This community can minimize ammonia accumulation in the water column through incorporation as bacterial biomass. Under suitable conditions (temperature, carbon:nitrogen ratio, pH, etc.), bacteria have a fast growth. Leonard et al. [30] estimated that the generation time for the free viable heterotrophic populations was around 2.5 h in laboratory conditions.

Heterotrophic bacteria utilize sugar, alcohol, and organic acids as energy source but exist in specialized species capable of decomposing cellulose, lignin, chitin, keratin, hydrocarbons, phenol, and other substances [31]. Heterotrophic bacteria are able to colonize a high diversity of environments; they are common in soil, freshwater, and saltwater. Aquatic environments are responsible to recycle high amounts of dissolved and particulate organic matter, playing one of the most important roles in the food webs [32]. In biofloc system, the heterotrophic bacteria colonize the feces, molts, dead organisms, and unconsumed food to produce bacterial biomass, which is consumed by detritivores [8]. Brown et al. [33] evaluated the biochemical compositions of seven strains of marine bacteria and reported protein content (dry weight) of 29–49%, carbohydrates 2.5–11.2, lipids 4–6%, and, additionally, the presence of all essential amino acids.

Chemoautotrophic bacterial community (i.e., nitrifying bacteria) obtains energy through oxidation of toxic nitrogen compounds. The nitrifying bacteria are naturally promoted by the presence of ammonia and nitrite as well as the accumulation of flocculated matter (used as substrate). The alkalinity consumed by these microorganisms must be replaced by different sources (i.e., sodium bicarbonate, calcium carbonate, or calcium hydroxide [34]). In laboratory conditions, the generation time of ammonia oxidizer bacteria was estimated to 25 h and nitrite oxidizer to 60 h [30].

The nitrifying bacteria thrive in a wide diversity of environments [35]. Besides the oxygen, toxic nitrogen compounds are the major concern into the biofloc systems. The main sources of ammonia are excretion of cultured organism and the decomposition of nonliving matter (dissolved and particulate). In BFT, three nitrogen conversion pathways occur for the removal of ammonia nitrogen: (a) photoautotrophic removal by algae, (b) autotrophic bacterial conversion from ammonia to nitrate, and (c) heterotrophic bacterial conversion of ammonia nitrogen directly to microbial biomass [36]. In long term, the most efficient process is the autotrophic, in which two bacterial groups are involved: (a) the ammonia-oxidizing bacteria, which obtain their energy by catabolizing unionized ammonia to nitrite, including the genera *Nitrosomonas*, *Nitrosococcus*, *Nitrosospira*, *Nitrosolobus*, and *Nitrosovibrio* and (b) the nitrite-oxidizing bacteria, which metabolize nitrite to nitrate, including the genera *Nitrobacter*, *Nitrococcus*, *Nitrospira*, and *Nitrospina* [37].
