WO2016019572A1

WO2016019572A1 - Mrna response-based variable flow control method and system for recirculating aquaculture system

Info

Publication number: WO2016019572A1
Application number: PCT/CN2014/083985
Authority: WO
Inventors: 阮贇杰; 朱松明
Original assignee: 浙江大学
Priority date: 2014-08-08
Filing date: 2014-08-08
Publication date: 2016-02-11

Abstract

Disclosed are an mRNA response-based variable flow control method and system of a recirculating aquaculture system, so as to achieve coordinated regulation of a biofilter of a recirculating aquaculture system and a microorganism response. The mRNA real-time expression quantity of functional microbial ammonia oxidizing bacteria (AOB) is obtained, so that the flow speed of the system is increased in an mRNA response high expression time period of the AOB, and the flow speed of the system is reduced in a response low expression time period. The problems are solved that an existing aquaculture water treatment biofilter is low in treatment efficiency and high in running energy consumption from the perspectives of coordinated regulation of the industrial aquaculture water treatment technology and a microorganism response.

Description

In particular, it relates to a method for producing a recirculating aquaculture system. Due to its resource conservation and environmental friendliness, the industrialized recirculating aquaculture system has become a model of aquaculture that is highly respected worldwide. Under the condition of ultra-high-density intensive culture, the core processing technology of the system is biological filtration. The main method is to configure the biofilter to complete the total ammonia nitrogen in the water body by using the microorganisms directed to the surface of the filter material. Total Total Ammonia Nitrogen) Removal with nitrite (ΝΌ ₂ -—N). The process is mainly carried out by the nitrification of the functional microbial group, and the total nitrogen nitrogen TAN is catalytically oxidized to hydroxylamine by ammonia monooxygenase of Ammonia Oxidizing Bacteria, and then oxidized by hydroxylamine to intermediate nitrite. Finally, the nitrite oxidizing bacteria: OB (Nitrite Oxidizing Bacteria) oxidizes nitrous acid into nitrate which is relatively harmless to the cultured object (]^ ₀₃ ^, and the full reaction rate-limiting step is Equation 1, and the relevant chemical formula is as follows:

NH ₃ + 0 ₂ + 2H ⁺ ■→ NH ₂ OH + H ₂ 0 1

N3⁄4OH + H ₂ 0 ― N0 ₂ - + 5H-2

NO ² " + C0 ₂ + 0.5 0 ₂ — NO ^3- 3 In the actual production practice, the system breeding scale (feeding amount of bait) and the effective volume of the biofilter are fixed, and the total processing performance of the biofilter is mainly related to the flow rate. In order to maintain the proper water quality conditions under high-density aquaculture load, the system operation process requires a high flow rate. Usually, the hydraulic retention time HRT (Hydraulic Retention Time) of the recirculating aquaculture system is 0 „5- lh, that is, the daily average biofilter Cycle 24 48 times.

However, the above-mentioned biofilter constant speed operation method still faces the following drawbacks: in the stable operation of the recirculating aquaculture system, the total ammonia nitrogen in the biofilter is mainly derived from the ammonia excretion of the culture object, and the relevant ammonia excretion rate is mainly related to the feed amount. In relation to feeding rhythm, the daily variation of water quality of the system shows a relative "peak-valley" concentration, thus maintaining a higher constant velocity flow of the system, although ensuring stable water quality, but during the peak period of pollutant concentration, the source of the biofilter matrix is due to the flow rate. Constantly limited, in the valley period, due to low base The mass concentration makes it inefficient operation, so the biological filter single cycle treatment efficiency is low. In order to achieve total pollutant throughput, the system needs to maintain a high flow rate, resulting in high overall energy consumption.

Since the water body of the recirculating aquaculture system is always in internal circulation, the pollutant concentration of the node at a certain time is the final result of the ammonia removal and biofilter treatment of the culture object. If the variable flow control is implemented based on this, the information source lags behind. Passively changing the system flow rate from the apparent level can not effectively improve the single treatment efficiency of the system biofilter, which relies on sacrificing system water quality to achieve energy saving.

The purpose of the present invention is to overcome the deficiencies of the prior art and provide a variable-flow control method and system for a recirculating aquaculture system based on an mRNA response, thereby realizing energy saving and consumption reduction of the circulating aquaculture system.

The invention detects the mRNA response expression of the picture A gene of the microbial AOB encoding ammonia monooxygenase in the biofilter internal filter of the circulating aquaculture system during the single feeding cycle, and extracts the system flow rate during the high expression period to Supply more matrix to its mRNA transcription, reduce flow rate during low expression periods to achieve energy savings. Since the mRNA encoding product of the amoA gene is ammonia monooxygenase (functional protein), its role is to catalyze the oxidation of total ammonia to hydroxylamine as a key mechanism for the rate-limiting step of nitrification, and the half-life of the protein is longer than the half-life of the mRNA. The flow rate of the expression segment can promote the effective accumulation of ammonia monooxygenase by the functional microorganism AOB, which can lay a material foundation for reducing the flow rate of the biofilter still has higher processing performance. According to the above principle, the present invention performs the precise regulation of the biological filter and the variable speed flow operation, and achieves the total flow reduction and energy saving of the circulating aquaculture system by improving its single treatment efficiency.

A variable-flow control method for recirculating aquaculture system based on mRNA response, synergistically regulating the biofilter and microbial response of the recirculating aquaculture system, obtaining the real-time expression of the mRNA of the functional microbe AOB, and increasing the system in the high expression period of the AOB mRNA response The flow rate, in response to low expression periods, reduces system flow rate.

The basic process for its implementation is:

1), monitoring the water quality of the system during the feeding cycle;

2), monitoring the expression level of the mRNA response of the microbial AOB of the filter material in the biofilter during the feeding cycle »

3), analyzing the high expression and low expression time period of functional microorganism AOB in the feeding cycle;

4). Comprehensive water quality data, increase the system flow rate by 10-30% in the lh period before and after the high expression peak of the functional microorganism AOB, and reduce the flow rate by 20-50% for the low expression period of mRNA.

The circulating aquaculture system mainly adopts a solid-liquid separation-biological purification two-stage treatment method to remove system pollutants, and the operation is stable. In the recirculating aquaculture system, the ratio of the bait dosage A (kg> to the effective volume B (m ³ ) of the biofilter in the single feeding cycle, A/B < 6.67.

The AOB mRNA response is based on high expression and low expression, and the ratio between the high expression value C and the low expression value D is C/D ≥ 5, or has a statistical difference ( Ρ < 0Λ ), the expression of the child unit The base copy number is 4ig RNA.

When the concentration of total ammonia nitrogen TAN (Tbtal Ammonia Nitrogen) in the system is ≥ 3.0 mg/L, the system operation does not decelerate regardless of the mR A response of the biofilter functional microorganism AOB.

A recirculating aquaculture system employing the method, using a variable speed flow control device.

The beneficial effect of the invention is that, by changing the constant flow rate strategy of the biological filter of the traditional recirculating aquaculture system, the mRNA expression response level of the functional microorganism AOB in the single feeding cycle is taken as a reference to increase the high expression segment flow rate and reduce the low mRNA expression, respectively. The section flow rate is a control measure to improve the efficiency of the single cycle of the biofilter. The invention overcomes the disadvantages of the operation of the traditional circulating aquaculture system, the extensive operation strategy of the biological filter and the high overall energy consumption, and has many advantages such as energy saving and efficiency improvement, and engineering application.

Fi diagram

1 is a flow chart showing an implementation of a variable speed flow control method for a recirculating aquaculture system based on an mRNA response.

As shown in FIG. 1, the present invention can be implemented by the following technical solutions:

1. The object of the present invention is a recirculating aquaculture system, which mainly relies on solid-liquid separation-biological purification two-stage treatment for system pollutant removal, and is stable in operation. The cultured biomass in the system has small fluctuations, and the feeding cycle and rhythm are fixed.

2. The water quality monitoring of the single-feeding cycle of the circulating water system is carried out at the beginning of the feeding time. The TAN content in the water is measured at intervals of 111. The sampling point is generally recommended to include at least 3 places (2 breeding ponds, 1 biofilter) ), the measurement duration is 24h, and the measurement method can adopt the national standard method such as the naphthalene reagent method, and the measurement accuracy is required to be 0.1 mg/L.

3. The expression of the mRNA response of the functional microorganism AOB in the biofilter is monitored by the following methods: 1. Sample preparation

(1) At the beginning of the feeding time, take 3 cells of the biofilter inner filter or 30 ml of the microbead filter at intervals of 1 h, and immediately add 5 ml of commercial RNA protection solution. (2) Place the sample on a magnetic oscillator for 10 minutes, centrifuge at high speed for 5 minutes on the centrifuge (operating conditions 4 Ό, 12000 rpm), discard the supernatant, and resuspend the biofilm mud sample to 2 ml of commercial RNA protection. Liquid, and stored in - 80 C for use.

2. Sample RN A extraction and reverse transcription cDNA (1) Sample RNA was extracted using a commercial RNA kit, and the procedure was carried out in accordance with the kit instructions. (2) The extracted total RNA was reverse transcribed into cDNA using a commercial kit, and the reaction condition was 42. (, lb; 70 ° C, 15 min; 8 ° C, + ∞.

III. Absolute quantification of mR A of functional microbial ammonia oxidizing bacteria AOB (Rt-qPCR)

(1) The amoA gene is the target region of the functional microbial ammonia oxidizing bacteria AOB. The amoA gene is used as the target region, and the selected species is amoA-1F /amoA-2R (GGGGTTTCTACTGGTGGT/

Amplification was carried out by CCCCTCKGSAAAGCCTTCTTC), and the PCR reaction conditions were: (i) 95. C, 5mm; (ii) 95°C, 20s; (iii) 55°C, 30s _; (iv) 72°C, 30s; (v) Repeat (ii)~(iv) steps for 40 cycles; (vi 72 ° C, 7 min; (vii) 8 ° C, + ∞. (2) The standard curve preparation method is; (i) ΙΟΟμΙ competent cells were added to the ΙΟμΙϋΝΑ solution, and placed on ice for 30 min. Then, after heating for 42 s for 45 s, Imin was placed in ice; (ii) 890 μl SOC medium was added and shake culture was carried out at 37 ° C for 60 min; (iii) ΙΟΟμΙ bacterial culture solution was uniformly coated on LB agar medium plate containing ampicillin. On, 37C was inverted and cultured overnight; (w) White colonies were selected, and the length of the insert in the vector was confirmed by PCR. (V) The cloning plasmid was extracted using a commercial plasmid extraction kit, and the plasmid concentration was determined by a micro-ultraviolet external spectrophotometer, and the labeling curve was prepared by using a gradient-diluted cloning plasmid. (3) Calculate the absolute abundance of the mRNA of the ammonia-oxidizing functional bacteria AOB of each sample by the standard curve, and the unit is (base copy number / g RNA:.

4. Identification of the expression level of AOB mRNA in the single feeding cycle

(1) The absolute expression level of the functional bacteria AOB monitored by each time node is plotted in a daily sequence to find the "high expression value" and "low expression value" on the curve. (2) Calculate the ratio between the high expression value C (base copy number / gRNA) and the low expression value D (base copy number / gRNA), if C / D ≥ 5, or have a unified learning difference (P <0.1), it is considered that the time point is a high expression value and a low expression value. Two or more consecutive high expression values or low expression values are considered to be high expression periods or low expression periods.

5. Biofilter flow rate adjustment based on mRNA response

(1) According to the comprehensive water quality data, the mR A high expression peak of the functional microorganism AOB increases the system flow rate by about 1030% in the lh period before and after, and decreases the flow rate by about 2050% in the low expression period of the mRNA. (2) When the total ammonia nitrogen concentration in the water in the system is ≥ 3.0 mg/L, the system does not decelerate regardless of the expression of the microbial mRNA response of the biofilter. According to the method of the present invention, an effect test was carried out on a recirculating aquaculture system in which the cultured object was Nile tilapia (Oreochromis Niloticus). Among them, the constant speed running time is 33 days, the variable speed running time is 36 days, and the variable speed control is realized by a frequency converter (VFD-015M). The effect is shown in Table 1. Table 1 Implementation effect of variable speed flow in actual circulating aquaculture system

Finally, it should be noted that the above embodiments are only for explaining the technical solutions of the present invention, and are not intended to be limiting; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art will understand that The technical solutions described in the foregoing embodiments are modified or equivalently substituted for some of the technologies; and the modifications and substitutions do not depart from the spirit and scope of the embodiments of the present invention.

Claims

A variable-flow control method for a recirculating aquaculture system based on an mRNA response, characterized in that the biofilter and microbial response of the recirculating aquaculture system are coordinated, and the functional microorganism AOB is obtained.

A response to high expression time period, increase system flow rate,

2. The method according to claim 1, wherein the basic flow of implementation is:

1), monitoring the water quality of the system during the feeding cycle;

2) monitoring the expression level of the mRNA response of the surface functional microorganism AOB in the biofilter inside the feeding cycle;

4). Comprehensive water quality data, increase the system flow rate by 10-30% for the high-expression peak of functional microbes AOB before and after the peak of mRNA, and reduce the flow rate by 20-50% for the low expression period of mRNA.

3. The method according to claim 1, wherein the circulating aquaculture system mainly adopts a solid-liquid separation-biological purification two-stage treatment method to remove system pollutants, and the operation is stable.

The method according to claim 3, characterized in that, in the recirculating aquaculture system, the ratio between the amount of feed A (kg) and the effective volume B (m ³ ) of the biofilter in a single feeding cycle , A/B < 6.67.

The method according to claim 1, wherein the mRNA of the AOB is in response to high expression and low expression, and the ratio between the high expression value C and the low expression value D is C/D ≥ 5, or has Statistical difference (P < 0.1), the expression value unit is base copy number / fig RNA.

6, according to the claims

The expression of AOB mRNA response did not slow down the system.

7. A recirculating aquaculture system employing the method of claim 1 wherein the means for controlling the flow rate is employed.