CN107941649A - The detection method of the inorganic nitrogen uptake rate of common microalgae in aquaculture pond - Google Patents
The detection method of the inorganic nitrogen uptake rate of common microalgae in aquaculture pond Download PDFInfo
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Abstract
The invention discloses a kind of detection method of the inorganic nitrogen uptake rate of common microalgae in aquaculture pond, step：(1) aquatic economic animal is measured from seedling is put to harvest section, inorganic nitrogen salt NaNO in pond3、NaNO2And NH4The concentration excursion of Cl；(2) according to minimum, maximum N concentration, seven N concentration gradients are designed；(3) using unazotized profound 107 13 nutrient solution of water as nutrient solution, by design, NaNO is used respectively3、NaNO2And NH4Nutrient solutions of the Cl with seven N concentration gradients, then corresponding addition15N inorganic nitrogen salts, make15N is the 10% of N mass, adds experiment algae；(4) cultivate；(5) micrometer algae weight, surveys δ15The weight percentage of N in N and microalgae；Inorganic nitrogen uptake rate is calculated again, present invention understands that the ecological environment residing for aquatic products, can accurately understand the source of nitrogen and whereabouts in microalgae, foundation is provided for aquatic products production process.
Description
Technical field
The present invention relates to stable isotope detection technique field, and common microalgae is inorganic more particularly in aquaculture pond
The detection method of nitrogen uptake rate.
Background technology
For a long time, due to lacking an overall technology, nitrogen is in the various flow paths of the land and water ecosystem and in food web
In contribution can not be by quantitative study.The research and development of stable nitrogen isotopes application aspect is rapid in recent years, it has also become Yi Zhongyou
The tracer means of effect, irreplaceable effect is played in animals and plants Physiological Ecology and environmental science.
The ecosystem experiment in add weight stable isotope (such as13C or15N), can follow the trail of source of students element source,
Whereabouts, process simultaneously analyze its contribution to food web and the ecosystem.By isotope labelling techniques obtain information be often
What routine techniques cannot provide, it is sometimes brand-new.This has just expanded the mankind and ecosystem structure, energy flow, material has been followed
Ring and the understanding to Human impact response.New foundation is provided to protect and recovering the injured ecosystem.
With China's rapid development of economy and the continuous improvement of living standards of the people, culture fishery develops rapidly, is
The people provide a large amount of low fat high-protein foods.But since aquaculture industry in China's does not become more meticulous still setting, putting seedling, bait throwing in
Etc. there is very big waste in a series of production processes, and make it that breeding water body eutrophication problem is serious.It is rich administering water body
Aquatic product quality is also taken into account during nutrient laden, so biological treating measure can only be utilized, and utilizes microalgae to stablize cultivation
Water body is one of best selection, and wherein nitrogen is one of key factor administered.Therefore, to common in aquaculture process
The monitoring of the nitrogen absorption rate of microalgae can effectively assess the health status of breeding water body.Therefore, it is necessary to develop one
Plant the detection method of the inorganic nitrogen uptake rate of common microalgae in more fast and accurately aquaculture pond.
The content of the invention
Nitrogen measure is time-consuming during the purpose of the present invention is overcoming breeding water body eutrophication control existing in the prior art
Long, manpower and materials consume the problem of big, there is provided the detection of the inorganic nitrogen uptake rate of common microalgae in a kind of aquaculture pond
Method.
Technical scheme is summarized as follows：
The detection method of the inorganic nitrogen uptake rate of common microalgae, includes the following steps in aquaculture pond：
(1) aquatic economic animal is measured from seedling is put to harvest section, inorganic nitrogen salt NaNO in pond3、NaNO2And NH4Cl
Concentration excursion；
(2) the minimum N concentration and maximum N concentration obtained according to step (1), designs seven N concentration gradients；
(3) using unazotized profound water 107-13 nutrient solutions for basis nutrient solution, according to the design of step (2), respectively with
NaNO3、NaNO2And NH4Cl is nitrogen source, configures the nutrient solution of seven N concentration gradients, then contains NaNO to each3Add in nutrient solution
Enter15N-NaNO3, make15N additive amounts are containing NaNO3The 10% of N mass in nutrient solution, contains NaNO to each2Added in nutrient solution15N-NaNO2, make15N additive amounts are containing NaNO2The 10% of N mass in nutrient solution, contains NH to each4Added in Cl nutrient solutions15N-
NH4Cl, makes15N additive amounts are containing NH4The 10% of N mass in Cl nutrient solutions, setting three is parallel, then is separately added into same concentrations
Experiment algae；
(4) sample that incubation step (3) obtains in illumination box, volume of culture 50mL, cultivation temperature are 28 DEG C,
Intensity of illumination is 39 μm of ol.m-2.s-1, incubation time 4h；
(5) with the glass fiber filter GF/C of dry constant weight, the algae solution that step (4) obtains is filtered respectively, is added 10mL and is gone
Ionized water rinses, and takes out the glass fiber filter GF/C with microalgae, doubling, with tinfoil bag after 60 DEG C of drying, constant weight, grindings
Wrap up in, freezen protective, the weight of the glass fiber filter GF/C of dry constant weight is weight 1；Dry the glass with microalgae of constant weight
The weight of fibrous filter membrane GF/C is the difference as gained microalgae weight of weight 2, weight 2 and weight 1, is surveyed using EA-IRMS systems
Determine δ15The weight percentage of N element in N and microalgae；
(6) absorption rate of the microalgae to inorganic nitrogen is calculated by the following formula：
Formula 1. in：V is absorption rate, its unit is：ug·g-1·h-1；
15Ns is algae sample after culture15N abundance；
15Nn is algae sample15The natural abundance of N, is 0.00365；
15Nenr is that culture medium is initial after adding tracer15N abundance, is 0.98；
T is incubation time, its unit is h；
PON is the total nitrogen contained by microalgae, its unit is ugg-1。
(formula 1. source：R.C.Dugdale,F.P.Wilkerson.The use of15N to measure
nitrogen uptake in eutrophic oceans；experimental considerations1,2[J]
.Limnology and oceanography,1986,31(4):673-689.)
Advantages of the present invention：Ecological environment residing for overall understanding aquatic products, including the environment of water body are relevant with other
Biology.Secondly, it can accurately understand the source of nitrogen and whereabouts in microalgae, foundation is provided for aquatic products production process.
Brief description of the drawings
Fig. 1 is absorption rate of the Oocystis Borgei to various concentrations nitrate nitrogen.
Fig. 2 is absorption rate of the Oocystis Borgei to various concentrations nitrite nitrogen.
Fig. 3 is absorption rate of the Oocystis Borgei to various concentrations ammonia nitrogen.
Embodiment
Below by specific embodiment, the present invention is further illustrated.
The present invention be using Oocystis Borgei as experiment algae, but this algae not to the present invention method be defined.Can be with
Using other algaes.
Prepare unazotized profound water 107-13 nutrient solutions；
K2HPO48mg；FeC6H5O7(1% solution) 0.2mg；Vitamin B1200μg；Vitamin B120.5~1.5 μ g；
NaHCO31g；Seawater 1000mL.
The detection method of the inorganic nitrogen uptake rate of common microalgae in 1. aquaculture pond of embodiment, including following step
Suddenly：
(1) aquatic economic animal (by taking Penaeus Vannmei as an example) is measured from seedling is put to harvest section (about 4 months), pond
Middle inorganic nitrogen salt NaNO3、NaNO2And NH4The concentration excursion of Cl；
(2) minimum N concentration and maximum N concentration (N concentration minimum value and the N in three inorganic nitrogen salts obtained according to step (1)
Concentration maxima), seven N concentration gradients are designed, are respectively 3.000mgL-1、6.000mg·L-1、13.000mg·L-1、
19.000mg·L-1、29.000mg·L-1、44.000mg·L-1、88.000mg·L-1；
(3) using unazotized profound water 107-13 nutrient solutions for basis nutrient solution, according to the design of step (2), respectively with
NaNO3、NaNO2And NH4Cl is nitrogen source, configures the nutrient solution of seven N concentration gradients, then contains NaNO to each3Add in nutrient solution
Enter15N-NaNO3, make15N additive amounts are containing NaNO3The 10% of N mass in nutrient solution, contains NaNO to each2Added in nutrient solution15N-NaNO2, make15N additive amounts are containing NaNO2The 10% of N mass in nutrient solution, contains NH to each4Added in Cl nutrient solutions15N-
NH4Cl, makes15N additive amounts are containing NH4The 10% of N mass in Cl nutrient solutions, setting three is parallel, then is separately added into same concentrations
(2.288×108cell·L-1) experiment algae Oocystis Borgei；
(4) sample that incubation step (3) obtains in illumination box, volume of culture 50mL, cultivation temperature are 28 DEG C,
Intensity of illumination is 39 μm of ol.m-2.s-1, incubation time 4h；
(5) with the glass fiber filter GF/C of dry constant weight, the algae solution that step (4) obtains is filtered respectively, is added 10mL and is gone
Ionized water rinses, and takes out the glass fiber filter GF/C with microalgae, doubling, with tinfoil bag after 60 DEG C of drying, constant weight, grindings
Wrap up in, freezen protective, the weight of the glass fiber filter GF/C of dry constant weight is weight 1；Dry the glass with microalgae of constant weight
The weight of fibrous filter membrane GF/C is the difference as gained microalgae weight of weight 2, weight 2 and weight 1, is surveyed using EA-IRMS systems
Determine δ15The weight percentage of N element in N and microalgae；
(6) absorption rate of the microalgae to inorganic nitrogen can be calculated by the following formula：
Wherein：V is absorption rate, its unit is：ug·g-1·h-1；
15Ns is algae sample after culture15N abundance；
15Nn is algae sample15The natural abundance of N, is 0.00365；
15Nenr is that culture medium is initial after adding tracer15N abundance, is 0.98；
T is incubation time, its unit is h, is 4h；
PON is the total nitrogen contained by microalgae, its unit is ugg-1。
(formula 1. source：R.C.Dugdale,F.P.Wilkerson.The use of15N to measure
nitrogen uptake in eutrophic oceans；experimental considerations1,2[J]
.Limnology and oceanography,1986,31(4):673-689.)
Sodium nitrate concentration gradient experiment result
Sodium nitrite concentration gradient experimental result
Ammonium chloride concentration gradient experiment result
The quantitative detection that the present invention absorbs inorganic nitrogen to be related to microalgae in aquaculture system provides technical support.For energy
The sorption enhanced of inorganic nitrogen in all kinds of cultivating pools is fast and accurately measured, assesses its public health risk, class where inventor
Topic group determines conversion rate of East Sea Island culture of Penaeus vannamei area microalgae in Zhanjiang to inorganic nitrogen with the detection method, and takes
Obtain good result.Solve the problems, such as nitrogen source and whereabouts unknown in aquaculture process.
As can be seen from Figure 1：Oocystis Borgei is 3.300mg in nitrogen concentration to the absorption rate of nitrate nitrogen in experiment
L-1~14.300mgL-1In the range of gradually rise, when nitrogen concentration is 3.300mgL-1When reach minimum, be
42.161ug·g-1·h-1, nitrogen concentration 14.300mgL-1When reach highest, be 73.173ugg-1·h-1, nitrogen concentration after
Height of continuing rising, the then trend (Fig. 1) that absorption rate presentation gradually reduces.One-way analysis of variance：F=228.279 ＞ F0.05(6,
14)=2.850, significant difference；Multiple range test shows significant difference between peak and other each groups.
As can be seen from Figure 2：Oocystis Borgei is in nitrogen concentration to the absorption rate of nitrite nitrogen in experiment
3.300mg·L-1When reach minimum, be 24.252ugg-1·h-1, with the rise of nitrogen concentration, absorption rate also rises therewith
Height, is 14.300mgL in nitrogen concentration-1When reach highest, be 81.120ugg-1·h-1, when nitrogen concentration continues rise, absorb
Speed, which is presented, gradually reduces trend (Fig. 2), one-way analysis of variance：F=192.511 ＞ F0.05(6,14)=2.850, difference is shown
Write；Multiple range test shows significant difference between peak and other each groups.
As can be seen from Figure 3：Oocystis Borgei is 96.800mgL in nitrogen concentration to the absorption rate of ammonia nitrogen in experiment-1
When reach minimum, be 20.856ugg-1·h-1, it is 14.300mgL in nitrogen concentration-1When reach highest, be 52.259ug
g-1·h-1, when nitrogen concentration is more than 20.900mgL-1When, drastically downward trend (Fig. 3), single factor test variance point is presented in absorption rate
Analysis：F=285.832 ＞ F0.05(6,14)=2.850, significant difference；Multiple range test is shown between first group, the 5th group and other groups
Significant difference.
The relation of planktonic algae and nutriment is complex, and its growth can be adjusted be subject to nutritive salt, to the profit of nitrogen source
With many physiology courses for being related to frustule, such as absorb, storage, and reduction, other chemical constituent (amino for synthesizing cell
Acid etc.) process.Many studies have shown that different types of unicellular alga is also different to the concentration requirement of nitrogen source.Certain
In the range of nutrient concentration, nutritive salt concentration of substrate is bigger, and seaweed is bigger to its absorption rate, its relation character closes enzymatic reaction
Kinetic curve.If nutrient concentration is too low, frustule division is slow, biomass is low, then can limit planktonic algae to battalion
Support the absorption of salt.Sufficient nutritive salt can promote the growth of planktonic algae, and then promote planktonic algae to the uptake rate
Rise, this is because the rising of nitrogen concentration makes concentration of substrate rise of the frond using a series of physiology courses of nitrogen in environment, because
And its assimilation increase within the specific limits, photosynthetic rate enhancing, algae, which absorbs, accelerates.However, excessive nutrition salt environment
Also inhibitory action can be produced to the absorption of microalgae, it may be possible to which water environment concentration has been over the energy of storage nutrition in frustule body
Power, therefore absorption rate has obvious reduction.
In the range of the nitrogen concentration of this experiment setting, different N concentration has significant shadow to the nutrient absorption of Oocystis Borgei
Ring.Absorption of the low nitrogen obviously to Oocystis Borgei is unfavorable, in the range of certain nitrogen concentration, absorption speed of the Oocystis Borgei to nitrogen
Rate increases with the rise of nutrient concentration；The concentration for testing Water Environment Nitrogen is 14.300mgL-1When, Oocystis Borgei reaches
It is respectively nitrate nitrogen 73.173ugg to its absorption maximum speed to nitrogen nutrition salt-1·h-1, nitrite nitrogen
95.872ug·g-1·h-1, ammonia nitrogen 52.560ugg-1·h-1；However, nitrogen of the excessive nutrition salt environment to Oocystis Borgei
Absorb produce inhibitory action, wherein, the inhibition of ammonia nitrogen clearly, when nitrogen concentration is more than 20.900mgL-1When, ammonia nitrogen
Drastically downward trend is presented in absorption rate.This experiment is consistent with other results of study.Author thinks, utilizes stable isotopic tracer
Method, microalgae is to the absorption rate of inorganic nitrogen in measure breeding water body that can be simple and quick, so as to judge the health of water body
Situation, direct evidence is provided to utilize microalgae to improve breeding water body eutrophication process in aquaculture process.Therefore, add
Stable isotope determination microalgae has very big application value to the method for the absorption rate of inorganic nitrogen in purifying aquaculture water.
Claims (1)
1. the detection method of the inorganic nitrogen uptake rate of common microalgae in aquaculture pond, it is characterized in that including the following steps：
(1) aquatic economic animal is measured from seedling is put to harvest section, inorganic nitrogen salt NaNO in pond3、NaNO2And NH4Cl's is dense
Spend excursion；
(2) the minimum N concentration and maximum N concentration obtained according to step (1), designs seven N concentration gradients；
(3) using unazotized profound water 107-13 nutrient solutions as basic nutrient solution, according to the design of step (2), respectively with NaNO3、
NaNO2And NH4Cl is nitrogen source, configures the nutrient solution of seven N concentration gradients, then contains NaNO to each3Added in nutrient solution15N-
NaNO3, make15N additive amounts are containing NaNO3The 10% of N mass in nutrient solution, contains NaNO to each2Added in nutrient solution15N-
NaNO2, make15N additive amounts are containing NaNO2The 10% of N mass in nutrient solution, contains NH to each4Added in Cl nutrient solutions15N-
NH4Cl, makes15N additive amounts are containing NH4The 10% of N mass in Cl nutrient solutions, setting three is parallel, then is separately added into same concentrations
Experiment algae；
(4) sample that incubation step (3) obtains in illumination box, volume of culture 50mL, cultivation temperature are 28 DEG C, illumination
Intensity is 39 μm of ol.m-2.s-1, incubation time 4h；
(5) with the glass fiber filter GF/C of dry constant weight, the algae solution that step (4) obtains is filtered respectively, adds 10mL deionizations
Water rinses, and takes out the glass fiber filter GF/C with microalgae, doubling, is wrapped up with tinfoil after 60 DEG C of drying, constant weight, grindings, is cold
Freeze and preserve, the weight of the glass fiber filter GF/C of dry constant weight is weight 1；Dry the filter of the glass fibre with microalgae of constant weight
The weight of film GF/C is the difference as gained microalgae weight of weight 2, weight 2 and weight 1, using EA-IRMS system measurements δ15N and
The weight percentage of N element in microalgae；
(6) absorption rate of the microalgae to inorganic nitrogen is calculated by the following formula：
Formula 1. in：V is absorption rate, its unit is：ug·g-1·h-1；
15Ns is algae sample after culture15N abundance,；
15Nn is algae sample15The natural abundance of N, is 0.00365；
15Nenr is that culture medium is initial after adding tracer15N abundance, is 0.98；
T is incubation time, its unit is h；
PON is the total nitrogen contained by microalgae, its unit is ugg-1。
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