CN107748237A - A kind of karst subterranean stream basin inertia organic carbon culture and detection method - Google Patents
A kind of karst subterranean stream basin inertia organic carbon culture and detection method Download PDFInfo
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Abstract
The present invention relates to a kind of karst subterranean stream basin inertia organic carbon culture and detection method, it is using karst subterranean stream basin water bodily indolence property organic carbon as research object, pass through indoor culture simulated experiment, test water body RDOC concentration and proportion, field data under authentic and valid reduction nature, and be combined with environmental data caused by carbonate rock weathering.It is main to solve Southwestern China karst area, it is changed into the problem in science that can form net carbon remittance after DOC in natural environment stored for extended periods with the presence or absence of DIC in inertia dissolved organic carbon, and Karst Geological Landscape mechanism in subterranean stream basin.
Description
Technical field
The present invention relates to karst carbon remittance stability study field, and in particular to a kind of karst subterranean stream basin inertia organic carbon
Culture and detection method.
Background technology
Carbon is one of most important biological element in the ecosystem.Wherein, organic carbon is in biogeochemical cycle and its
Importantly role (Meybeck M are play in the biogeochemical cycle of his trace element；Et al, 1999), study river
In organic carbon to terrestrial ecosystems tool be of great significance.Riverine organic carbon both include humus, lipid, polysaccharide,
The relatively stable organic matter such as polypeptide and colloidal substance, includes the unstable Yi Beixi such as amino acid and carbohydrate again
The organic matter that bacteria microorganism utilizes (is otherwise known as " active material ") (SchlesingerW H；et al,1981).Generally, with grain
Organic carbon of the footpath less than 0.45 μm is dissolved organic carbon (dissolvedorganic carbon, DOC), and particle diameter is more than 0.45 μm
Organic carbon is particulate organic carbon (particle organic carbon, POC) (ShenY；et al,2015).Water body is dissolved with
Machine carbon (DOC) is one of organic carbon pool maximum in the world, and its total amount is roughly equivalent to CO in global atmosphere2Phosphorus content, often
In organic carbon about 0.4Pg of the year by terrestrial ecosystems by covalency ocean disposal, wherein about 60% is DOC, 40% is POC,
By river terrestrial ecosystems Global Terrestrial Ecosystem net primary productivity is roughly equivalent to the organic carbon of ocean disposal
1%~2% (SpitzyA；et al,1991).DOC composition is sufficiently complex, and only 10%~20% water body is organic at present
Thing can determine as certain specific component, therefore the general general content with organic carbon represents the concentration of water body organism
(Dawson R；et al.1981).Planktonic bacteria is both the analyst and conversion person of aquatic ecosystem nutriment, is had again
The circulation of matter energy and memory function (Madoni P, et al；2007).A considerable amount of DOC can be absorbed profit by them
With, and after ingest through protozoic and enter classical food chain (Azam F；et al.2007).Miniature organism passes through photosynthetic work
It is metabolized with, heterotrophism, virolysis effect and protozoal predation etc., DOC components can be brought to redistribute, dissolved organic carbon
(Jiao N Z are converted from activity to inertia；At el.2011), so as to continuously produce the inertia for being difficult to be used again
Dissolved organic carbon (Recalcitrant dissolved organic carbon, RDOC), makes water body organic carbon in long-time chi
Degree (millennial scale) stores (JiangY that becomes a reality；et al.2013).The fixation of organic carbon, DIC can be accelerated again to organic
The process of carbon conversion, and then Karst Geological Landscape effect partial carbon is converged and be stored in water body (JiangY for a long time in the form of RDOC；et
Al.2013), its stability greatly enhances.In ocean, it is impossible to which it is 95% strong to account for DOC for the inertia organic carbon being bioavailable
(Jiao Nianzhi；Deng 2011), it can be seen that, in the case where planktonic bacteria acts on, caused RDOC is the important composition portion of water body carbon cycle
Point.The property and flux and geochemical process of research water body inertia organic carbon are to clear and definite carbon distribution characteristics and karst carbon
Stability of converging has vital effect.And there has been no the method for testing of the clear and definite karst aquifer inertia organic carbon of scholar at present.
The content of the invention
Up to the present, people gradually recognize that Karst Geological Landscape effect is not purely inorganic geological process, and biological agent is extensive
Participate.Wherein, water body DOC is changed into RDOC by planktonic bacteria in a manner of its unique biological metabolism, so as to store for a long time
In water body, the Stable Carbon for forming long time scale converges.Nowadays, the research to RDOC is concentrated mainly on " marine microorganism carbon
This scientific research field of pump ", and focus mainly on RDOC generation process and mechanism.But accounting for the land area of China 1/3
Karst area, water body inertia organic carbon are not probed into emphatically but.The technical problems to be solved by the invention are to provide a kind of karst
Subterranean stream basin inertia organic carbon culture and detection method, it is using karst subterranean stream basin water bodily indolence property organic carbon as research pair
As, by indoor culture simulated experiment, test water body RDOC concentration and proportion, under authentic and valid reduction nature
Field data, and be combined with environmental data caused by carbonate rock weathering.Mainly solve in Southwestern China karst area,
Can be changed into subterranean stream basin with the presence or absence of DIC in inertia dissolved organic carbon, and Karst Geological Landscape mechanism after DOC
The problem in science of net carbon remittance is formed in natural environment stored for extended periods.
The technical scheme that the present invention solves above-mentioned technical problem is as follows：A kind of karst subterranean stream basin inertia organic carbon culture
With detection method, including the steps：
Step S1, prepare following utensil：If metal tweezers, metal cutting nippers, Brown Glass Brown glass bottles and jars only are some, the blue cover glass bottle of spiral
Do, core filter is some, silica bead is some, graduated cylinder is some, masking foil is some, filter hole is respectively 3 μm, 0.45 μm and 0.22 μm
Polyether sulfone parent's water system miillpore filter is some, polytetrafluoroethylsample sample bottle is some and marking pen；
Step S2, to metal tweezers, metal cutting nippers, Brown Glass Brown glass bottles and jars only, spiral indigo plant cover glass bottle, core filter, silicon
Pearl and graduated cylinder are cleaned；
Step S3, to Brown Glass Brown glass bottles and jars only, spiral indigo plant cover glass bottle, core filter, silica bead and polyether sulfone it is hydrophilic be micro-
Hole filter membrane carries out sterilization treatment, standby；
Step S4, study and subterranean stream exit is selected in area as subterranean stream water sample sample point, by 0.45 μm of polyether sulfone
Close water system miillpore filter is assemblied on core filter, is filtered in the presence of pump is filtered by vacuum by the hydrophilic system's micropore of polyether sulfone
Membrane filtration water sample measures 250ml filtered fluids by graduated cylinder and poured into spiral indigo plant cover glass bottle, tighten lid in core filter
The body of spiral indigo plant cover glass bottle is wrapped up after son and with masking foil, re-records sample number, and 500ml water is obtained in same sample point
Sample, loaded in polytetrafluoroethylsample sample bottle；Remaining sample point follows above-mentioned sampling work in research area, until field sampling is complete
Into, altogether obtain T sample；
Step S5, the water sample that each sample point is taken is mixed and is well mixed in a vessel, then by being equipped with 3 μm
The Suction filtration device pre-filtering mixed sample of polyether sulfone parent's water system miillpore filter, remove the large granular impurity in mixed sample and big grain
Footpath planktonic organism, is once filtered mixed sample；Again by being equipped with the suction filtration of 0.22 μm of polyether sulfone parent's water system miillpore filter
Water sample is once filtered in device filtering, obtains secondary filter mixed sample, 0.22 μm of hydrophilic system of polyether sulfone is gripped with metal tweezers
Miillpore filter, then shredded with cutting nippers, it is positioned in Brown Glass Brown glass bottles and jars only, while silica bead is poured into Brown Glass Brown glass bottles and jars only and is covered in
On broken film, and T × 5ml secondary filter water sample is added in Brown Glass Brown glass bottles and jars only, bottle cap is sealed and is put into CHA-S gas baths perseverance
Shaken 2 hours in warm oscillator；After concussion terminates, Brown Glass Brown glass bottles and jars only 30 minutes are stood, take supernatant liquid to be protected as inoculation liquid
Stay；
Step S6, the spiral indigo plant cover glass bottle of one is taken, to its inside addition 250mL through 0.45 μm of hydrophilic system of polyether sulfone
The 2mmol/L calcium bicarbonate solutions of filtering with microporous membrane, add sterilizing suction nozzle using the pipettor that maximum range is 1000 μ L, to this
Spiral indigo plant cover glass bottle adds after 2.5ml inoculation liquids and wraps up shading with masking foil, as the following inoculation sample water of test each time
Negative control value during body dissolved organic carbon concentration；
2.5ml inoculation liquids are added according to above-mentioned same mode, 2.5mL inoculation liquids is drawn successively and is transferred to and fills filtering
In each spiral indigo plant cover glass bottle of liquid, after each sample is well mixed with inoculation liquid, then takes in spiral indigo plant cover glass bottle and connect
Kind of sample 25ml, its water body dissolved organic carbon initial concentration is tested by the analyzers of Multi N/C 3100, after cover spiral is blue
Vial capping is closed and wrapped up with masking foil, is put into progressive culture in 18 DEG C of constant incubators；
Step S7, tested every 15 days using identical method in a spiral indigo plant cover glass bottle in incubation and be inoculated with sample
The water body dissolved organic carbon concentration of product, original place will be placed in spiral indigo plant cover glass bottle after the completion of test, be continued using the same terms
Culture, until water body dissolved organic carbon concentration not untill changing, and this water body dissolved organic carbon concentration is closest to inertia
Dissolved organic carbon concentration, therefore the dissolved organic carbon concentration no longer changed is defined as the organic concentration of carbon of water body inertia.
The beneficial effects of the invention are as follows：The culture of inertia dissolved organic carbon and test are incorporated into karst subterranean stream water first
In body, the existence form of carbon during perfect karst carbon cycle；Water sample is trained in the case where height reduces natural environmental condition
Support, obtain the actual RDOC concentration of karst aquifer to greatest extent, can directly solve karst subterranean stream basin and whether there is
RDOC and RDOC contents are how many problem in science, so as to prove that Karst Geological Landscape effect has net carbon remittance effect.
On the basis of above-mentioned technical proposal, the present invention can also do following improvement：
Further, the blue lid of metal tweezers, metal cutting nippers, Brown Glass Brown glass bottles and jars only, spiral is cleaned in step S2 in the following manner
Vial, core filter, silica bead and graduated cylinder：
Metal tweezers and metal cutting nippers first dip in absolute ethyl alcohol using absorbent cotton and are wiped repeatedly, and are all removed to spot；Horse is used again
Not 450 DEG C of stove calcination 2 hours, surface organic carbon residual and bacterium residual are removed, it is standby with masking foil parcel after cooling；
All Brown Glass Brown glass bottles and jars onlys, spiral indigo plant cover glass bottle, core filter, silica bead and graduated cylinder are immersed in weight successively
Potassium chromate 200g, concentrated sulfuric acid 3.6L, pure water 400mL chromic acid lotion acid cylinder in, and pull out, then use after reflecting 12 hours respectively
Each utensil after a large amount of running water cleaning and dippings, it is inverted water until the utensil of cleaning and flows out untill its wall do not hang droplet, so
Multipass is cleaned successively with pure water and ultra-pure water again afterwards, finally dries each utensil in 110 DEG C of baking ovens.
It is using the above-mentioned further beneficial effect of scheme：Every utensil is cleaned before culture, removes removal of residue
Accomplish to prevent to introduce other pollutants, ensure to obtain in natural environment the authentic and valid of the organic concentration of carbon of inertia under microbial action
Value.
Further, in step S3 in the following manner to Brown Glass Brown glass bottles and jars only, spiral indigo plant cover glass bottle, core filter,
Silica bead and polyether sulfone parent water system miillpore filter carry out sterilization treatment：By all Brown Glass Brown glass bottles and jars onlys, spiral indigo plant cover glass bottle, core
Filter, silica bead and polyether sulfone parent's water system miillpore filter are put into high-pressure sterilizing pot, in 0.1MPa pressure or kettle temperature
Sterilized 25 minutes for constant temperature at 120 DEG C, sterilizing preserves each utensil closed sterile soft after terminating.
It is using the above-mentioned further beneficial effect of scheme：Inertia organic carbon is dense under microbial action in acquisition natural environment
The key of the authentic and valid value of degree is to prevent the intrusion of external miscellaneous bacteria, and every utensil sterilization treatment is micro- in natural environment to obtain
The authentic and valid value of the organic concentration of carbon of inertia under biological agent.
Brief description of the drawings
Fig. 1 is dissolved organic carbon dynamic detection figure.
Embodiment
The principle and feature of the present invention are described below in conjunction with instantiation, example is served only for explaining this hair
It is bright, it is not intended to limit the scope of the present invention.
A kind of karst subterranean stream basin inertia organic carbon culture and detection method, including the steps：
Step S1, prepare following utensil：To complete RDOC culture with detecting, it is necessary to which the utensil and purposes got all the ready include：
Metal tweezers, for absorbing filter membrane；Metal cutting nippers, for shredding filter membrane；Brown Glass Brown glass bottles and jars only (100ml), for shaking and holding
Inoculation liquid；Spiral indigo plant cover glass bottle (500ml), for cultivating water sample, until experiment is completed；Core filter (1000ml),
For filtering water sample, water body impurity and filtering inoculation liquid are removed；Polyether sulfone parent's water system miillpore filter (50mm；3μm/0.45μm/
0.22 μm), for retaining microorganisms in water, make inoculation liquid；Polytetrafluoroethylsample sample bottle (500ml), for preserving water sample；Silicon
Pearl, for shaking filter membrane；Graduated cylinder (250ml), measures filtrate；Masking foil, for shading；Marking pen, for written contents；
2mmol/L calcium bicarbonate solutions, compareed for recessiveness.
Step S2, to obtain the authentic and valid value of the organic concentration of carbon of inertia under microbial action in natural environment, test it
Preceding needs are accomplished to prevent to introduce other pollutants.Therefore, need to be cleaned in every utensil before culture, remove removal of residue.
Metal tweezers, metal cutting nippers, Brown Glass Brown glass bottles and jars only, spiral indigo plant cover glass bottle, core filter, silica bead are cleaned in the following manner
And graduated cylinder：
Metal tweezers and metal cutting nippers first dip in absolute ethyl alcohol using absorbent cotton and are wiped repeatedly, and are all removed to spot；Horse is used again
Not 450 DEG C of stove calcination 2 hours, surface organic carbon residual and bacterium residual are removed, it is standby with masking foil parcel after cooling；
All Brown Glass Brown glass bottles and jars onlys, spiral indigo plant cover glass bottle, core filter, silica bead and graduated cylinder are immersed in weight successively
Potassium chromate 200g, concentrated sulfuric acid 3.6L, pure water 400mL chromic acid lotion acid cylinder in, and pull out, then use after reflecting 12 hours respectively
Each utensil after a large amount of running water cleaning and dippings, it is inverted water until the utensil of cleaning and flows out untill its wall do not hang droplet, so
Multipass is cleaned successively with pure water and ultra-pure water again afterwards, finally dries each utensil in 110 DEG C of baking ovens；
Step S3, the key for obtaining the authentic and valid value of the organic concentration of carbon of inertia under microbial action in natural environment is anti-
The only intrusion of external miscellaneous bacteria, therefore every utensil is both needed to sterilization treatment.In the following manner to Brown Glass Brown glass bottles and jars only, spiral indigo plant lid glass
Glass bottle, core filter, silica bead and polyether sulfone parent water system miillpore filter carry out sterilization treatment：By all Brown Glass Brown glass bottles and jars onlys,
Spiral indigo plant cover glass bottle, core filter, silica bead and polyether sulfone parent's water system miillpore filter are put into high-pressure sterilizing pot,
0.1MPa pressure or kettle temperature are that constant temperature sterilizes 25 minutes at 120 DEG C, and sterilizing preserves each utensil closed sterile soft after terminating,
It is standby；
Step S4, study and subterranean stream exit is selected in area as subterranean stream water sample sample point, by 0.45 μm of polyether sulfone
Close water system miillpore filter is assemblied on core filter, is filtered in the presence of pump is filtered by vacuum by the hydrophilic system's micropore of polyether sulfone
Membrane filtration water sample measures 250ml filtered fluids by graduated cylinder and poured into spiral indigo plant cover glass bottle, tighten lid in core filter
The body of spiral indigo plant cover glass bottle is wrapped up after son and with masking foil, re-records sample number, to make culture experiment go back to greatest extent
Former actual karst subterranean stream basin nature no light conditions, blake bottle need shading treatment.And obtain 500ml water in same sample point
Sample, loaded in polytetrafluoroethylsample sample bottle；Remaining sample point follows above-mentioned sampling work in research area, until field sampling is complete
Into, altogether obtain T sample；
Step S5, the water sample that each sample point is taken is mixed and is well mixed in a vessel, then by being equipped with 3 μm
The Suction filtration device pre-filtering mixed sample of polyether sulfone parent's water system miillpore filter, remove the large granular impurity in mixed sample and big grain
Footpath planktonic organism, is once filtered mixed sample；
Water sample is once filtered in the Suction filtration device filtering by being equipped with 0.22 μm of polyether sulfone parent's water system miillpore filter again, obtains
Secondary filter mixed sample, because planktonic bacteria particle diameter is all higher than 0.22 μm, now 0.22 μm of polyether sulfone parent's water system miillpore filter
Most planktonic bacterias in water sample can be retained, and maintain original structure of community.0.22 μm of polyethers is gripped with metal tweezers
Sulfone parent's water system miillpore filter, then shredded with cutting nippers, it is positioned in Brown Glass Brown glass bottles and jars only, while silica bead is poured into Brown Glass Brown glass bottles and jars only
In be covered on broken film, and T × 5ml secondary filter water sample is added in Brown Glass Brown glass bottles and jars only, bottle cap is sealed and is put into
Shaken 2 hours in CHA-S gas bath constant temperature oscillators；
Step S6, after concussion terminates, Brown Glass Brown glass bottles and jars only 30 minutes are stood, takes supernatant liquid to retain as inoculation liquid, connects
Kind liquid bacterial quorum sensing keeps constant, and planktonic bacteria abundance is original 100 times；The another spiral indigo plant cover glass bottle for taking one, to
Its internal 2mmol/L calcium bicarbonate solution through the hydrophilic system's filtering with microporous membrane of 0.45 μm of polyether sulfone for adding 250mL, using most
Wide range is that 1000 μ L pipettor adds sterilizing suction nozzle, and masking foil is used after adding 2.5ml inoculation liquids to the spiral indigo plant cover glass bottle
Wrap up shading.In 2mmol/L calcium bicarbonate solution, Ca2+And HCO3-In karst aquifer concentration range, and organic carbon content
For 0mg/L, no external source germ contamination, the moon during sample water body dissolved organic carbon concentration can be inoculated with as following test each time
Property control value.Instrument test result can all have an error, negative control value in theory should 0, if instrument is measured not to be 0 to be exactly
Have error, that is, need with the inoculation sample water body dissolved organic carbon concentration measured subtract negative control value be only accurately measurement knot
Fruit.
2.5ml inoculation liquids are added according to above-mentioned same mode, 2.5mL inoculation liquids is drawn successively and is transferred to and fills filtering
In each spiral indigo plant cover glass bottle of liquid, because bacterial growth breeding needs corresponding carbon source, nitrogen source, inorganic salts, pH, Oxygen Condition
And particular nutrient, and contain material base and life condition needed for bacterium in each sample point filtered fluid, and it is original to ensure
Living environment is not changed, and other nutriments are no longer added into blake bottle.Each sample is well mixed with inoculation liquid
Afterwards, inoculation sample 25ml in spiral indigo plant cover glass bottle is first taken, its water body is tested by the analyzers of Multi N/C 3100 and is dissolved with
Machine carbon initial concentration, after by spiral indigo plant cover glass bottle be capped close and wrapped up with masking foil, be put into 18 DEG C of constant incubators
Row culture；
Step S7, tested every 15 days using identical method in a spiral indigo plant cover glass bottle in incubation and be inoculated with sample
The water body dissolved organic carbon concentration of product, original place will be placed in spiral indigo plant cover glass bottle after the completion of test, be continued using the same terms
Culture, until water body dissolved organic carbon concentration not untill changing, and this water body dissolved organic carbon concentration is closest to inertia
Dissolved organic carbon concentration, therefore the dissolved organic carbon concentration no longer changed is defined as the organic concentration of carbon of water body inertia.
A complete experimental study process is described below：
In June, 2016 is studied 10 sample points of hair village subterranean stream basin, be designated as respectively MC, WD, TC, DY, XL,
LL、BY、SG、SW、BD.Required utensil and medicine specifically have：Metal tweezers one；Metal cutting nippers one；Brown Glass Brown glass bottles and jars only
(60ml) one；11, spiral indigo plant cover glass bottle (500ml)；Core filter (1000ml) is a set of；Aperture is 3 μm of polyether sulfones
Close water system miillpore filter 5 is opened, and 0.45 μm of polyether sulfone parent water system miillpore filter 11 is opened, 0.22 μm of polyether sulfone parent water system miillpore filter 2
；10, polytetrafluoroethylsample sample bottle (500ml)；One, graduated cylinder (250ml)；Masking foil is a roll of；Marking pen two；2mmol/L
Calcium bicarbonate solution 1000ml.
Each sample point uses 0.45 μm of the hydrophilic system's filtering with microporous membrane water sample of polyether sulfone, is measured by graduated cylinder
250ml filtered fluids are poured into spiral indigo plant cover glass bottle, are tightened lid and are wrapped up body with masking foil, record sample number.It is each in addition
Individual sample point respectively takes 500ml water samples to be loaded in polytetrafluoroethylsample sample bottle, and takes back laboratory.
500ml water samples acquired by each sampling point are well mixed in laboratory, first pass through the hydrophilic system's micropore filter of 3 μm of polyether sulfones
Film, obtain a filtered fluid.Filtered fluid is passed through into the hydrophilic system's filtering with microporous membrane of 0.223 μm of polyether sulfone again, obtained secondary
Filtered fluid, and obtain the water system miillpore filter for retaining total planktonic bacteria.In the super-clean bench of laboratory total float will be retained using cutting nippers
The water system miillpore filter of trip bacterium, which shreds, to be put into brown sample bottle, and 50mL secondary filter liquid is added in Brown Glass Brown glass bottles and jars only,
Add after about 15g silica beads, Brown Glass Brown glass bottles and jars only bottle is put into CHA-S gas bath constant temperature oscillators and shaken 2 hours.Wait to shake
After end, Brown Glass Brown glass bottles and jars only 30 minutes are stood, take 2.5mL upper liquids to be added separately to 10 spiral indigo plant cover glass sample bottles successively
In.The another 2mmol/L calcium bicarbonate solutions through 0.45 μm of filtering with microporous membrane for taking spiral indigo plant cover glass bottle to add 250mL, together
Sample adds 2.5ml inoculation liquids.Aforesaid operations step is completed in the super-clean bench of laboratory, ensures the pollution of no external miscellaneous bacteria.
After well mixed, 25mL liquid is taken from sample bottle, is placed in the analyzers of Multi N/C 3100 and determines, obtain organic carbon training
Support initial concentration value.Spiral indigo plant cover glass sample bottle is placed in original place after the completion of test, continues to cultivate using the same terms.Culture
During every 15 days test water body dissolved organic carbon concentration and record obtain table one.As shown in figure 1, and painted according to table one
Dissolved organic carbon dynamic detection figure processed.
In sample culturing 0-15 days, biological community structure will not change in blake bottle, and nutriment is sufficient, micro-
Biology can show impregnable nature, continue vital movement, and carbon sequestration bacterium changes the DIC in culture water sample
It is organic carbon so as to improving water body DOC concentration.But as the extension of incubation time, the effect of carbon sequestration bacterium start to weaken,
Water body organic carbon is shown based on consumption, and DOC concentration in the case where microorganism is using conversion is begun to decline.It is organic to cultivate latter stage dissolving
Concentration of carbon fluctuation is smaller and no longer reduces, and no longer DOC concentration is had an impact in this stage microbial action, it can be considered that
This part organic carbon is that microorganisms in water is relatively difficult by inertia dissolved organic carbon.
The culture sample RDOC contents of table 1
The foregoing is only presently preferred embodiments of the present invention, be not intended to limit the invention, it is all the present invention spirit and
Within principle, any modification, equivalent substitution and improvements made etc., it should be included in the scope of the protection.
Claims (3)
1. a kind of karst subterranean stream basin inertia organic carbon culture and detection method, it is characterised in that including the steps：
Step S1, prepare following utensil：Metal tweezers, metal cutting nippers, Brown Glass Brown glass bottles and jars only are some, the blue cover glass bottle of spiral is some,
Core filter is some, silica bead is some, graduated cylinder is some, masking foil is some, filter hole is respectively 3 μm, 0.45 μm and 0.22 μm
Polyether sulfone parent's water system miillpore filter is some, polytetrafluoroethylsample sample bottle is some and marking pen；
Step S2, to metal tweezers, metal cutting nippers, Brown Glass Brown glass bottles and jars only, spiral indigo plant cover glass bottle, core filter, silica bead and
Graduated cylinder is cleaned；
Step S3, Brown Glass Brown glass bottles and jars only, spiral indigo plant cover glass bottle, core filter, silica bead and the hydrophilic system's micropore of polyether sulfone are filtered
Film carries out sterilization treatment, standby；
Step S4, study and subterranean stream exit is selected in area as subterranean stream water sample sample point, 0.45 μm of polyether sulfone is hydrophilic
It is that miillpore filter is assemblied on core filter, passes through polyether sulfone parent's water system miillpore filter mistake in the presence of pump is filtered by vacuum
Drainage sample measures 250ml filtered fluids by graduated cylinder and poured into spiral indigo plant cover glass bottle, after tightening lid in core filter
And the body of spiral indigo plant cover glass bottle is wrapped up with masking foil, sample number is re-recorded, and 500ml water samples are obtained in same sample point,
Loaded in polytetrafluoroethylsample sample bottle；Remaining sample point follows above-mentioned sampling work in research area, until field sampling is completed,
T sample is obtained altogether；
Step S5, the water sample that each sample point is taken is mixed and is well mixed in a vessel, then by being equipped with 3 μm of polyethers
The Suction filtration device pre-filtering mixed sample of sulfone parent's water system miillpore filter, removes the large granular impurity in mixed sample and big particle diameter floats
Trip biology, is once filtered mixed sample；Again by being equipped with the Suction filtration device of 0.22 μm of polyether sulfone parent's water system miillpore filter
Water sample is once filtered in filtering, obtains secondary filter mixed sample, the hydrophilic system's micropore of 0.22 μm of polyether sulfone is gripped with metal tweezers
Filter membrane, then shredded with cutting nippers, it is positioned in Brown Glass Brown glass bottles and jars only, while silica bead is poured into Brown Glass Brown glass bottles and jars only and is covered in broken film
On, and T × 5ml secondary filter water sample is added in Brown Glass Brown glass bottles and jars only, bottle cap is sealed and is put into CHA-S gas bath constant temperature and is shaken
Swing in device and shake 2 hours；After concussion terminates, Brown Glass Brown glass bottles and jars only 30 minutes are stood, take supernatant liquid to retain as inoculation liquid；
Step S6, the spiral indigo plant cover glass bottle of one is taken, to its inside addition 250mL through the hydrophilic system's micropore of 0.45 μm of polyether sulfone
The 2mmol/L calcium bicarbonate solutions of membrane filtration.Add sterilizing suction nozzle using the pipettor that maximum range is 1000 μ L, to the spiral
Blue cover glass bottle adds after 2.5ml inoculation liquids and wraps up shading with masking foil, molten as the following inoculation sample water body of test each time
Solve negative control value during organic concentration of carbon；
According to above-mentioned same mode, 2.5mL inoculation liquids are drawn successively and are transferred to each spiral indigo plant lid glass for filling filtered fluid
In glass bottle, after each sample is well mixed with inoculation liquid, then inoculation sample 25ml in spiral indigo plant cover glass bottle is taken, passed through
The analyzers of Multi N/C 3100 test its water body dissolved organic carbon initial concentration, after by spiral indigo plant cover glass bottle capping closing simultaneously
Wrapped up with masking foil, be put into 19 DEG C of constant incubators and cultivated；
Step S7, tested every 15 days using identical method in incubation and sample is inoculated with a spiral indigo plant cover glass bottle
Water body dissolved organic carbon concentration, original place will be placed in spiral indigo plant cover glass bottle after the completion of test, continues to cultivate using the same terms,
Until water body dissolved organic carbon concentration not untill changing, and this water body dissolved organic carbon concentration is dissolved with closest to inertia
Machine concentration of carbon, therefore the dissolved organic carbon concentration no longer changed is defined as water body inertia dissolved organic carbon concentration.
2. karst subterranean stream basin inertia organic carbon culture according to claim 1 and detection method, it is characterised in that step
Metal tweezers, metal cutting nippers, Brown Glass Brown glass bottles and jars only, spiral indigo plant cover glass bottle, core filtering dress are cleaned in rapid S2 in the following manner
Put, silica bead and graduated cylinder：
Metal tweezers and metal cutting nippers first dip in absolute ethyl alcohol using absorbent cotton and are wiped repeatedly, and are all removed to spot；Muffle furnace is used again
450 DEG C of calcinations 2 hours, surface organic carbon residual and bacterium residual are removed, wrapped up after cooling with masking foil standby；
All Brown Glass Brown glass bottles and jars onlys, spiral indigo plant cover glass bottle, core filter, silica bead and graduated cylinder are immersed in dichromic acid successively
Potassium 200g, concentrated sulfuric acid 3.6L, pure water 400mL chromic acid lotion acid cylinder in, and respectively reflect 12 hours after pull out, then with largely
Each utensil after running water cleaning and dipping, it is inverted water until the utensil of cleaning and flows out untill its wall do not hang droplet, Ran Houzai
Multipass is cleaned successively with pure water and ultra-pure water, finally dries each utensil in 110 DEG C of baking ovens.
3. karst subterranean stream basin inertia organic carbon culture according to claim 1 and detection method, it is characterised in that step
In the following manner to Brown Glass Brown glass bottles and jars only, spiral indigo plant cover glass bottle, core filter, silica bead and the hydrophilic system of polyether sulfone in rapid S3
Miillpore filter carries out sterilization treatment：By all Brown Glass Brown glass bottles and jars onlys, spiral indigo plant cover glass bottle, core filter, silica bead and gather
Ether sulfone parent's water system miillpore filter is put into high-pressure sterilizing pot, and constant temperature sterilizes at being 120 DEG C in 0.1MPa pressure or kettle temperature
25 minutes, sterilizing preserved each utensil closed sterile soft after terminating.
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