WO2002030835A1

WO2002030835A1 - A process for nitrifying water in closed system hatcheries of penaeid and non penaeid prawn

Info

Publication number: WO2002030835A1
Application number: PCT/IN2000/000097
Authority: WO
Inventors: Cini Achutan; Rosamma Philip; I. S. Bright Singh
Original assignee: SCHOOL OF ENVIRONMENTAL STUDIES; Department of Biotechnology of Ministry of Science and Technology India
Current assignee: SCHOOL OF ENVIRONMENTAL STUDIES; Department of Biotechnology of Ministry of Science and Technology India
Priority date: 2000-10-09
Filing date: 2000-10-09
Publication date: 2002-04-18
Anticipated expiration: 2003-04-09
Also published as: KR20020092350A; KR100751198B1; JP2004511329A

Abstract

A process for nitrifying water in closed system hatcheries of penaeid and non penaeid prawn which comprises in a first step of treating water with an ammonia oxidizing consortia to convert the ammonium present in water of the hatchery system to NO2 and in a second step of nitrite oxidizing with a nitrite oxidizing consortia for converting NO2 into NO3.

Description

TITLE OF THE INVENTION

A process for nitrifying water in closed system hatcheries of penaeid and non penaeid prawns.

BACKGROUND OF INVENTION In prawn hatcheries unionized ammonia, >0.1 ppm at alkaline pH(>7.5) is toxic to the larvae. In closed larval rearing systems, NH, /NH-, is accumulated as the excretory product of nitrogen metabolism of prawn larvae and also as the product of ammonification of faeces and left over feed. It s generally recognized that the presence of such ammonia in prawn hatcheries contributes to the toxicity. The hatchery systems known in the art may be the penaeid hatchery systems having a salinity optima at around 30 ppt or a non penaeid hatchery system having a salinity optima at around 13 ppt.

OBJECTS OF THE INVENTION

An object of this invention is to propose a process for nitrifying water in closed system hatcheries of penaeid and non penaeid prawns.

Another object of this invention is to propose a process for nitrifying water in closed system hatcheries of penaeid and non penaeid prawns which is simple and at the same time efficient.

Still another object of this invention is to propose a bioreactor for nitrifying water in closed system hatcheries of penaeid and non penaeid prawns.

A further object of this invention is to propose a bioreactor for nitrifying water in closed system hatcheries of penaeid and non penaeid prawns which may be employed in the larval tank itself.

A still further object of this invention is to propose a bioreactor for nitrifying water in closed system hatcheries of penaeid and non penaeid prawns which is employed external to the hatchery system.

DESCRIPTION OF INVENTION

According to this invention there is provided a process for nitrifying water in closed system hatcheries of penaeid and non penaeid prawn which comprises in a first step of treating water with an ammonia oxidizing consortia to convert the ammonium present in water of the hatchery system to NO,-, and in a second step of nitrite oxidizing with a nitrite oxidizing consortia for converting NO,, into NO-,.

Basically two kinds of organisms such as 1. Ammonia oxidizing (NH, ~>N0„ and 2. Nitrite oxidising (NO -^N0„) bacteria are required for the process and bioreactors of this invention. Just like any other organisms nitrifiers also have salinity optima and therefore both ammonia and nitrite oxidizers having salinity optima at around 30 ppt have been developed for the bioreactors meant for penaeid hatchery systems and nitrifying consortia having salinity optima at around 13 ppt have been developed for the bioreactor meant for non penaeid hatchery systems. Such oxidizers are:

1. AMOPCU-1: Ammonia oxidising consortia meant for penaeid culture system.

2. NIOPCU-1: Nitrite oxidising consortia meant for penaeid culture system.

3. AMONPCU-1: Ammonia oxidising consortia meant for non- penaeid culture system.

4. NIONPCU-1: Nitrite oxidising consortia meant for non- penaeid culture system.

The consortia are composed of chemolithotrophic and mixotrophic nitrifiers which oxidize ammonia and nitrite to nitrite and nitrate respectively. Associated with them are heterotrophic bacteria which live on exudates of the nitrifiers. Such constitutents were not segragated, as their interactions were essential for obtaining highest nitrifying potential and better performance.

OPTIMUM GROWTH REQUIREMENTS OF THE CONSORTIA.

Optimum pH, temperature, substrate concentration and salinity of these oxidizers have been determined as they are essential for their mass production in fermentor and also for immobilisation in the reactor support material.

Nitrifying Consortia Optimum growth requirements

pH Temperature Salinity(ppt) Substrate o-. μg.mL

AMOPCU-1 7.5 28 30 10

NIOPCU-1 7.0 37 25 10

AMONPCU-1 8.5 28 10 10

NIONPCU-1 7.0 37 15 10 MASS PRODUCTION OF NITRIFYING CONSORTIA IN FERMENTOR

The nitrifying consortia can be amplified as per the requirements supplying the above optimum growth conditions.

The amplification can be started from a 10%(v/v) starter culture. The substrate as (NH,)₂SO, and NaN0₂ are added as small aliquots as they are being consumed so as to

+ — maintain a level not less than 10 ppm NH, -N or N0_? -N.

The fermentor vessel is covered with black cloth to prevent photoinactivation of nitrifiers. It takes 25 to 30 days for attaining stationary phase characterised by cessation of substrate (NH, -N or NO -N) uptake and product (NO,, -N and N0--N) formation. Profound wall growth is seen in all cases and they are scraped off and the entire culture drained off from the fermentor, pH adjusted, substrate added to optimum and maintained at 4 C either in glass or polythene bottles. Whenever the substrate is depleted and pH alteration occurred they have to be adjusted manually.

UNIT NITRIFYING ACTIVITY (UNA) OF THE CONSORTIA)

One- unit nitrifying activity (UNA) of a nitrifying consortium is defined as the quantity of nitrifying biomass which can bring about the generation of lμ mole N0„- N

-1 -1 min L in the case of ammonia oxidizers (UNAa) and the consumption of lμ mole N0_?- N min -1L-1 in the case of nitrite oxidizers (UNAn) under optimum conditions. This is the most appropriate method of quantifying the potential of any nitrifying consortium, to evaluate it and to assess how much quantity of the culture should be used to charge a reactor for obtaining the expected activity.

DETERMINATION OF QUANTITY OF CONSORTIA The appropriate medium is prepared with optimum pH, and substrate concentration in triplicate in 500 mL screw capped bottles (7.5cm O.D and 21cm ht) and maintained at the optimum temperature in a serological water bath. Filter sterilized (by passing air either from air compressor or air blower through a pipeline membrane filter device) air

-1 at a rate of lL.min is passed through an air sparger immersed in the medium. The bottles are inoculated with varying aliquots (1,5,10,15 mL) of the consortium. Evaporation loss is compensated by adding sterile distilled water. Once in two hours substrate utilization and product formation are measured for a period of 24 hours. Based on the value, the total number of units available with lmL aliquots of the consortium can be determined. From this the requirement of the quantity of consortium for one reactor also can be worked out and it is this volume that is to be used for immobilizing in the reactor bed.

EXAMPLE 1

Suppose lmL aliquot of the given AMOPCU-1 contains 10 UNAa, Capability of the consortium is: lUNAa: Production of lμ mole N0„- N min -1L-1 from

NH.⁺-N lμ mole: 14 ⁴.0 jtig N0₂~ N min-1L-1 lOμ mole: 14x10 u.min^-1L^-1 That means, one mL aliquot of the consortium can produce

140μg 0₂- N min -1L-1 the medium which contains lOμg NH, +-

N mL^_1 (10,000 μg NH^ ^L^-1 ) . This is the measure of capability of the consortium which can consume the entire quantity of NH, +-N(10,000μgL-1 ) with in 71.43 minutes under optimum conditions.

SUPPORT MATERIAL FOR IMMOBILIZING NITRIFYING BACTERIA Considering various factors such as inertness in aquatic system, light weight, hydrophobicity , easiness to mould in to any shape and easy availability, plastic has been selected as the basic material for the support. Beads of, for example, 5.0 mm diameter with a hole of 2.00 mm diameter at the centre and with sparkings on the surface are moulded out of high density polyethylene (HDPE) , low density polyethylene (LDPE) , polyvinylchloride (PVC), polypropylene (PP) , polystyrene (PS) , polycarbonate (PC), nylon and ABS and screened against each consortium. Considering the effectiveness in immobilizing cells, cost and easiness to mould the following type of plastics were selected for each consortium.

NAME OF THE CONSORTIUM TYPE OF PLASTIC AMOPCU-1 Polystyrene(PS)

NIOPCU-1 Low density polyethylene (LDPE) AMONPCU-1 Polystyrene(PS)

NIONPCU-1 Low density polyethylene (LDPE)

DESCRIPTION OF INVENTION WITH REFERENCE TO ACCOMPANYING DRAWINGS

Further object and advantages of this invention will be more apparent from the ensuing description when read in conjunction with the accompanying drawings and wherein: Fig.l. Shows the outer shell of an in-situ stringed bed suspended reactor (SBSBR), Fig.2. Shows the inner cartridge of a SBSBR, Fig.3. Shows an ex-situ packed bed bioreactor (PBBR) Fig.4. Shows two PBBR reactors in series.

In accordance with this invention, the drawings illustrate two types of reactor. A first type of reactor shown in Figs.l and 2 is an In-situ strined bed suspended bioreactor (SBSBR) for use in the larval rearing tank itself during the process of larval rearing, and the second type namely an Ex-situ packed bed bioreactor (PBBR) is meant for nitrifying, fresh seawater before using for larval rearing and spent water after completion of the larval cycle, so that, the water may be recirculated is shown in Figs.3 & 4. The reactors may be employed for both penaeid (Penaeus indicus and Penaeus monodon) and non penaeid (Macrobrachium rosenbergii) prawns.

Referring to Figs. 1 and 2, the SBSBR reactor comprises on outer shell OS made of for example, fibreglass and has an inlet Wl for introduction of water. An inner cartridge IC is disposed within- shell OS. The reactor is kept suspended 1 foot below the water level on a float. Fig.2 illustrates the inner cartridge IC made of a plastic, such as perspex. Inner cartridges IC has an air lift pump LP. When air lift pump LP is operated water enters the reactor through holes PI on top wall TP of the shell and passes through the cartridge IC and comes out through the central pipe or air lift pump LP. The frame work of the inner catridge IC is made in such a way that larvae, plankton and food particles when entering the reactor passes out through air lift pump LP without mutilation and damage. The reactor has holes P₂ through which the string bead SB with the oxidizer immobilized thereon passes and as shown. Ex-situ packed bed bioreactor (PBBR)

The reactor of Fig.3 is made of fibre glass shell SH mounted

2 on a base SB of for example 30 cm and an overall height of 45cm. The base plate SB is perforated and has, for example, 9 PVC pipes LP of 2cm diameter, 10 cm equidistance which is placed on a PVC support PS positioned 5cm above the base. An outlet pipe OP emerges from the base of the reactor and bends upwardly. Each pipe LP mounted on the perforated plate SB functions as an air lift pump when air is passed through. Each zone surrounding the air lift pump can be designated as an aeration cell when packed with the plastic beads PB with the oxidizer immobilizer thereon selected for each consortium and positioned through holes P„. Nine such aeration cells can be operated as by way of example. The reactor is filled to the top of the airlift pump with beads suitable for the consortium used.

The ex situ PBBR may have a water storage facility and with a recirculatory system, as shown in Fig.4. The reactor of Fig.4 has overhead tank (A) which opens to a tank at ground level (C). From the bottom of the tank (C) an outlet pipe (D) connects to the first reactor Fl (with ammonia oxidizers). An outlet pipe from this reactor connects to the second reactor (F2) which is with nitrite oxidizers. This reactors empties into the collection tank (K) from where it can be pumped back to the overhead tank or used for larval rearing. The reactor F., and F₂ are to standard size and the total number of such reactors required for each hatchery depends on the volume of water used per season.

^In Fi -⁴ reactors F and F₂ are placed in between the tank C and K kept at ground level. There is, therefore, gravitational flow of water from the overhead tank A to tank C and successively to the reactors and finally to the collecting tank K. Energy needed for the operation is only to pump water (either spent water or fresh sea water after disinfection and salinity adjustments) to the overhead tank. In case nitrification is not completed by just one circulation/passage through the system, it can be recirculated through the treatment system over and again. But this recirculation can be effectively avoided if the rate of flow of water in the system is regulated in such a way that a prolonged hydraulic retention period is provided.

Tanks A, C and K may, if required be of uniform size and made of for example fibreglass. The tank C serves as aeration basin also where degradation of organics can take place provided the water is adequately aerated.

One of the important advantages is that different types filters for the removal of particulate matter and UV water disinfection gadgets may also be connected on line with the bioreactor as required without any modification of the system.

The Ex situ PBBR has for example 9 aeration cells, through each one of which air can be passed, providing immense flexibility in the overall capability of the system.

+ —

Thus, if the contents of NH, -N and N0₂ -N in the water to be nitrified are more, it would be just sufficient to increase the quantity of air passed through the air lift pumps. This wil enhance the rate of circulation of water and increase the contact time with the nitrifiers immobilized on beads, besides, the hydraulic retention time also can be regulated by regulating the flow rate to allow more contact period.

Another advantage of the reactor is that when used for penaeid prawns can be converted very easily for non penaeids by just replacing the reactor with the immobilised AMOPCU- 1 and NIOPCU-1 with the ones immobilised with AMONPCU-l and NIONPCU-1 meant for non-penaeid systems.

ACTIVATION OF THE REACTORS

a. In situ stringed bed suspended bioreactor.

The bioreactors are placed in the activation mode in an activation system. This consists of a serological water bath set at the optimum temperature of the consortium used. The top lid of the reactors are removed and covered with transparent aerosol arresters made in perspex. The reactors are filled with autoclaved sea water with the required

+ — salinity and supplemented with lOppm NH, -N and N0„ -N respectively. Reactors are charged with the required units of nitrifying consortium to obtain the desired activity

(the quantity of inoculum is determined based on UNA of the consortium and the minimum nitrifying potential required per reactor). The air lift pump is operated by passing air from a compressor at the rate of 1L per minute. Once

+ — in 24 hours the consumption of NH, -N and N0„ -N build up of N0„ -N and NO-, N in both ammonia and nitrite oxidizing reactors respectively are determined along with adjustment of pH. In accordance with the removal, the substrates are added and the activation continued for 72 hours. By this period the nitrifying consortia will have adsorbed on to the support material. Now the reactors are ready enough to be moved on to the filed. The reactors on reaching the site are allowed to hang on the float. The air tubing is connected to the main air supply of the hatchery and the air flow rate is regulated at the rate of 1L per minute.

b. Ex-situ packed bed reactor.

The reactors (F₁ and F_?) are filled with seawater in the appropriate salinity supplemented with lOppm NH, -N and N0« -N. The air lift pumps are operated and an aliquot of 20 L consortia (ammonia oxidising and nitrite oxidising respectively in the reactors F₁ and F„) are inoculated and the operation continued till the nitrification is established. Subsequently circulation of water is established by opening the valve of the overhead tank and the flow rate regulated to give sufficient hydraulic retention for attaining complete nitrification.

Claims

WE CLAIM:

1. A process for nitrifying water in closed system hatcheries of penaeid and non penaeid prawn which comprises in a first step of treating water with an ammonia oxidizing consortia to convert the ammonium present in water of the hatchery system to N0„ and in a second step of nitrite oxidizing with a nitrite oxidizing consortia for converting N0₂ into NO.,.

2. A process as claimed in claim 1 wherein the ammonia oxidizing consortia for penaeid culture system is different to that of a non-penaeid culture system.

3. A process in claimed claim 1 wherein the nitrite oxidizing consortia for a penaeid culture system is different to that of a non-penaeid culture system.

4. A process as claimed in claim 1 wherein the consortia is immobilized on a support.

5. A process as claimed in claim 4 wherein said support comprises plastic beads.

6. An apparatus for nitrifying water in closed system hatcheries of penaeid and non-penaeid prawns comprising a reactor having an outer shell and an inner cartridge, at least one pipe being an air lift pump, said inner cartridge havin an ammonia or nitrite oxidizing consortia for penaeid and non-penaeid culture systems.

7. An apparatus as claimed in claim 6 comprising a plurality of strands or strings of beads held within said reactor, the consortia immobilized on said beads.

8. An apparatus as claimed in claim 7 wherein said beads are of plastic such as polystyrene or low density polyethylene.

9. An apparatus as claimed in claim 6 wherein said inner cartridge has a plurality of spaced slits for allowing water to flow therethrough, and a plurality of spaced holes for said strands or strings of beads to pass therethrough.

10. An apparatus for nitrifying water in closed system hatcheries of penaeid and non penaeid prawns comprising a reactor having a base with a plurality of pipes extending upwardly therefrom, said pipes being air pipes an ammonia or nitrite oxidizing consortia disposed within said reactor, an outlet connected to said base for discharge of the treated water.

11. An apparatus as claimed in claim 10 said ammonia or nitrite oxidizing consortia is embedded in beads, strands or strings of said beads extending within said reactor.

12. An apparatus as claimed in claims 10 and 11 comprising an overhead tank connected to a lower tank, the outlet of said lower tank connected to a first reactor having an ammonia oxidizer therein, said first reactor connected to a second reactor having a nitrite oxidizer therein, the outlet from said second reactor connected to a collecting tank and from where water is pumped to said overhead tank.

13. A process for nitrifying water in closed system hatcheries of penaeid and non penaeid prawns substantially as herein described and illustrated.

14. An apparatus for nitrifying water in closed system hatcheries of penaeid and non penaeid prawns substantially as herein described and illustrated.