MD4465C1 - Process for determining the dehydrogenase activity in fermentation biomass - Google Patents
Process for determining the dehydrogenase activity in fermentation biomass Download PDFInfo
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- MD4465C1 MD4465C1 MDA20140089A MD20140089A MD4465C1 MD 4465 C1 MD4465 C1 MD 4465C1 MD A20140089 A MDA20140089 A MD A20140089A MD 20140089 A MD20140089 A MD 20140089A MD 4465 C1 MD4465 C1 MD 4465C1
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- optical density
- dehydrogenase activity
- tff
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- 238000000034 method Methods 0.000 title claims abstract description 30
- 230000000694 effects Effects 0.000 title claims abstract description 29
- 101710088194 Dehydrogenase Proteins 0.000 title claims abstract description 27
- 239000002028 Biomass Substances 0.000 title claims abstract description 16
- 238000000855 fermentation Methods 0.000 title claims abstract description 11
- 230000004151 fermentation Effects 0.000 title claims abstract description 10
- 239000000203 mixture Substances 0.000 claims abstract description 24
- 230000003287 optical effect Effects 0.000 claims abstract description 21
- 238000011534 incubation Methods 0.000 claims abstract description 20
- 239000000243 solution Substances 0.000 claims abstract description 17
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 11
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 11
- 238000000605 extraction Methods 0.000 claims abstract description 11
- 239000008103 glucose Substances 0.000 claims abstract description 11
- 238000004364 calculation method Methods 0.000 claims abstract description 10
- YYGNTYWPHWGJRM-UHFFFAOYSA-N (6E,10E,14E,18E)-2,6,10,15,19,23-hexamethyltetracosa-2,6,10,14,18,22-hexaene Chemical compound CC(C)=CCCC(C)=CCCC(C)=CCCC=C(C)CCC=C(C)CCC=C(C)C YYGNTYWPHWGJRM-UHFFFAOYSA-N 0.000 claims abstract description 8
- BHEOSNUKNHRBNM-UHFFFAOYSA-N Tetramethylsqualene Natural products CC(=C)C(C)CCC(=C)C(C)CCC(C)=CCCC=C(C)CCC(C)C(=C)CCC(C)C(C)=C BHEOSNUKNHRBNM-UHFFFAOYSA-N 0.000 claims abstract description 8
- PRAKJMSDJKAYCZ-UHFFFAOYSA-N dodecahydrosqualene Natural products CC(C)CCCC(C)CCCC(C)CCCCC(C)CCCC(C)CCCC(C)C PRAKJMSDJKAYCZ-UHFFFAOYSA-N 0.000 claims abstract description 8
- 229940031439 squalene Drugs 0.000 claims abstract description 8
- TUHBEKDERLKLEC-UHFFFAOYSA-N squalene Natural products CC(=CCCC(=CCCC(=CCCC=C(/C)CCC=C(/C)CC=C(C)C)C)C)C TUHBEKDERLKLEC-UHFFFAOYSA-N 0.000 claims abstract description 8
- PKDBCJSWQUOKDO-UHFFFAOYSA-M 2,3,5-triphenyltetrazolium chloride Chemical compound [Cl-].C1=CC=CC=C1C(N=[N+]1C=2C=CC=CC=2)=NN1C1=CC=CC=C1 PKDBCJSWQUOKDO-UHFFFAOYSA-M 0.000 claims abstract description 7
- 238000006356 dehydrogenation reaction Methods 0.000 claims abstract description 6
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims abstract description 5
- BEIHVSJTPTXQGB-QIXACUJNSA-N n'-anilino-n-phenyliminobenzenecarboximidamide Chemical compound C=1C=CC=CC=1N\N=C(C=1C=CC=CC=1)\N=NC1=CC=CC=C1 BEIHVSJTPTXQGB-QIXACUJNSA-N 0.000 claims abstract description 5
- 239000008055 phosphate buffer solution Substances 0.000 claims abstract description 5
- 238000004458 analytical method Methods 0.000 claims description 8
- 238000012937 correction Methods 0.000 claims description 6
- 238000011088 calibration curve Methods 0.000 claims description 5
- 239000003153 chemical reaction reagent Substances 0.000 claims description 5
- 238000005259 measurement Methods 0.000 claims description 4
- 238000013019 agitation Methods 0.000 claims description 3
- 239000007853 buffer solution Substances 0.000 claims description 3
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 2
- 235000019800 disodium phosphate Nutrition 0.000 claims description 2
- 229910000397 disodium phosphate Inorganic materials 0.000 claims description 2
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical class [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims 1
- 238000002156 mixing Methods 0.000 abstract 1
- 239000000523 sample Substances 0.000 description 28
- 235000019441 ethanol Nutrition 0.000 description 7
- 239000002994 raw material Substances 0.000 description 4
- 238000012360 testing method Methods 0.000 description 3
- 230000001133 acceleration Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 230000001143 conditioned effect Effects 0.000 description 2
- 238000004821 distillation Methods 0.000 description 2
- 210000003608 fece Anatomy 0.000 description 2
- VLKZOEOYAKHREP-UHFFFAOYSA-N methyl pentane Natural products CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- VMGAPWLDMVPYIA-HIDZBRGKSA-N n'-amino-n-iminomethanimidamide Chemical compound N\N=C\N=N VMGAPWLDMVPYIA-HIDZBRGKSA-N 0.000 description 2
- 230000036284 oxygen consumption Effects 0.000 description 2
- BOLDJAUMGUJJKM-LSDHHAIUSA-N renifolin D Natural products CC(=C)[C@@H]1Cc2c(O)c(O)ccc2[C@H]1CC(=O)c3ccc(O)cc3O BOLDJAUMGUJJKM-LSDHHAIUSA-N 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- LNOBZXNCABUBKK-UHFFFAOYSA-N 2,3,5-triphenyltetrazolium Chemical compound C1=CC=CC=C1C(N=[N+]1C=2C=CC=CC=2)=NN1C1=CC=CC=C1 LNOBZXNCABUBKK-UHFFFAOYSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 238000003975 animal breeding Methods 0.000 description 1
- 230000003851 biochemical process Effects 0.000 description 1
- 230000000035 biogenic effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 238000004040 coloring Methods 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
- 238000005265 energy consumption Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000000696 methanogenic effect Effects 0.000 description 1
- -1 mono-substituted sodium phosphate Chemical class 0.000 description 1
- 235000019799 monosodium phosphate Nutrition 0.000 description 1
- 238000011158 quantitative evaluation Methods 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 239000010865 sewage Substances 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
Invenţia se referă la biotehnologie, şi anume la un procedeu de determinare a activităţii dehidrogenazei în biomasă la fermentare. The invention relates to biotechnology, namely to a method for determining the activity of dehydrogenase in biomass during fermentation.
Invenţia poate fi aplicată pentru controlul expres al procesului de fermentare anaerobă a materiei prime organice în tehnologia de biogaz şi pentru dirijarea acestui proces. Activitatea sumară a fermenţilor dehidrogenezici reprezintă un indicator al activităţii biologice generale în procesul de fermentare anaerobă, care caracterizează activitatea microorganismelor însuşi, determină viteza şi profunzimea proceselor biochimice din bioreactor. The invention can be applied for the express control of the process of anaerobic fermentation of organic raw material in biogas technology and for directing this process. The summary activity of the dehydrogenase enzymes is an indicator of the general biological activity in the anaerobic fermentation process, which characterizes the activity of the microorganisms themselves, determines the speed and depth of the biochemical processes in the bioreactor.
Este cunoscut procedeul de control al procesului de fermentare anaerobă a materiei prime organice, care constă în determinarea analitică a materiei prime organice, care include determinarea analitică a indicatorilor de consum chimic al oxigenului (CCO) şi consum biochimic (CBO) al oxigenului în biomasa supusă fermentării, pH-ului şi a temperaturii în bioreactor. Însă acest procedeu este indirect, nu permite evaluarea cantitativă a activităţii procesului de metanogeneză şi, respectiv, controlul operativ al formării biogazului pe parcursul proceselor anaerobe de fermentare a biomasei [1]. The control procedure of the anaerobic fermentation process of the organic raw material is known, which consists in the analytical determination of the organic raw material, which includes the analytical determination of the chemical oxygen consumption (CCO) and biochemical oxygen consumption (CBO) indicators in the subjected biomass fermentation, pH and temperature in the bioreactor. But this procedure is indirect, it does not allow the quantitative evaluation of the activity of the methanogenesis process and, respectively, the operative control of biogas formation during the anaerobic processes of biomass fermentation [1].
Cel mai apropiat conform esenţei tehnice şi rezultatului scontat este procedeul de determinare a activităţii dehidrogenazei, care include introducerea în proba analizată de biomasă supusă fermentării a soluţiilor de glucoză şi 2,3,5-trifenil-tetrazoliu clorurat (TTC), incubarea amestecului în condiţii de vid cu extragerea ulterioară cu alcool şi determinarea colorimetrică a densităţii optice a soluţiei colorate de trifenil-formazan (TFF) în calitate de produs al reacţiei de dehidrogenare a TTC [2]. The closest according to the technical essence and the expected result is the procedure for determining the dehydrogenase activity, which includes the introduction of glucose and 2,3,5-triphenyl-tetrazolium chlorinated (TTC) solutions into the analyzed biomass sample subjected to fermentation, the incubation of the mixture in conditions of vacuum with the subsequent extraction with alcohol and the colorimetric determination of the optical density of the colored solution of triphenyl-formazan (TFF) as a product of the dehydrogenation reaction of TTC [2].
Dezavantajele acestui procedeu constau în durata mare a reacţiei, volumul mare de muncă şi capacitatea înaltă de absorbţie a energiei, lipsa factorilor de stabilizare a activităţii dehidrogenazei (extractele colorate se decolorează repede) şi inconvenienţa determinării şi măsurării activităţii dehidrogenazei în cazul unui număr mare de probe analizate, ceea ce îl face nerentabil din punct de vedere economic. Totodată, procesul de incubare, având loc timp de 60 min, este îndelungat. The disadvantages of this procedure are the long reaction time, the high volume of work and the high energy absorption capacity, the lack of factors stabilizing the dehydrogenase activity (colored extracts quickly discolor) and the inconvenience of determining and measuring the dehydrogenase activity in the case of a large number of samples analyzed, which makes it unprofitable from an economic point of view. At the same time, the incubation process, taking place for 60 min, is long.
Problema soluţionată prin prezenta invenţie constă în accelerarea, reducerea volumului de muncă a procesului de analiză şi ieftinirea procedeului de efectuare a analizei. The problem solved by the present invention consists in accelerating, reducing the workload of the analysis process and making the analysis process cheaper.
Procedeul de determinare a activităţii dehidrogenazei în biomasă la fermentare prevede introducerea în proba analizată a soluţiei de glucoză şi de clorură de 2,3,5-trifeniltetrazoliu (TTC), incubarea amestecului în condiţii de vid, extragerea ulterioară cu alcool, determinarea colorimetrică a densităţii optice a soluţiei colorate de trifenilformazan (TFF) formate în rezultatul reacţiei de dehidrogenare a TTC cu calcularea ulterioară a activităţii dehidrogenazei, totodată în componenţa amestecului supus analizei se introduce scualenă, în cantitate de (5,0…5,5)10-4 % în raport cu volumul amestecului analizat, şi soluţie tampon de fosfat cu pH-ul 7,2, totodată se utilizează soluţii de 0,2 M de glucoză şi 2% TTC, procesul de incubare având loc în condiţii mezofile la temperatura de 33±1°C timp de 25…30 min, cu agitarea ulterioară prin scuturare timp de 5 min, iar calcularea activităţii dehidrogenazei se efectuează conform formulei: The procedure for determining the activity of dehydrogenase in biomass during fermentation provides for the introduction of glucose and 2,3,5-triphenyltetrazolium chloride (TTC) solution into the analyzed sample, incubation of the mixture under vacuum conditions, subsequent extraction with alcohol, colorimetric determination of density optical images of the colored solution of triphenylformazan (TFF) formed as a result of the dehydrogenation reaction of TTC with the subsequent calculation of the dehydrogenase activity, at the same time squalene is introduced into the composition of the mixture under analysis, in an amount of (5.0...5.5)10-4 % in relation to the volume of the analyzed mixture, and phosphate buffer solution with pH 7.2, at the same time solutions of 0.2 M glucose and 2% TTC are used, the incubation process taking place in mesophilic conditions at a temperature of 33±1 °C for 25...30 min, with subsequent agitation by shaking for 5 min, and the calculation of the dehydrogenase activity is carried out according to the formula:
, ,
unde where
- activitatea dehidrogenazei, mg TFF/ml·h; - dehydrogenase activity, mg TFF/ml·h;
- valoarea absolută a densităţii optice a probei analizate, media a 3 măsurări; - the absolute value of the optical density of the analyzed sample, the average of 3 measurements;
- valoarea absolută a densităţii optice a probei sterile; - the absolute value of the optical density of the sterile sample;
- valoarea absolută a densităţii optice a reactivilor fără probă; - the absolute value of the optical density of reagents without sample;
t- timpul incubării, h; t- incubation time, h;
- coeficient de corecţie a densităţii optice conform curbei de calibrare, mg TFF/ml; - optical density correction coefficient according to the calibration curve, mg TFF/ml;
- raportul dintre volumul amestecului pentru extragere în probă şi volumul probei pentru calibrare. - the ratio between the volume of the mixture for extraction in the sample and the volume of the sample for calibration.
Soluţia tampon, utilizată la realizarea procedeului, conţine fosfat de sodiu monosubstituit (NaH2PO4) - 68,4 g/L şi disubstituit (Na2HPO4) - 31,6 g/L. The buffer solution, used to carry out the procedure, contains mono-substituted sodium phosphate (NaH2PO4) - 68.4 g/L and di-substituted (Na2HPO4) - 31.6 g/L.
Rezultatul tehnic obţinut în urma realizării invenţiei propuse constă în accelerarea procesului de incubare datorită prezenţei în componenţa amestecului a microadaosului de scualenă (2,6,10,15,19,23 - hexametiltetracoza - 2,6,10,14,18,22 - hexaen), care reduce durata de adaptare a microorganismelor la noile substraturi şi asigură o efectuare mai rapidă a reacţiei de dehidrogenare a TTC şi de formare a TFF. Utilizarea unor concentraţii mai mari de glucoză şi TTC duce la accelerarea suplimentară a reacţiei ţintă de dehidrogenare. Agitarea prin scuturare a amestecului după incubare îl omogenizează şi îmbunătăţeşte solubilitatea şi extragerea TFF pentru determinarea colorimetrică a densităţii optice a probei. După intensitatea colorării se apreciază cantitatea de formazan, care este proporţională cu activitatea dehidrogenazei. The technical result obtained following the realization of the proposed invention consists in accelerating the incubation process due to the presence in the composition of the microaddition of squalene (2,6,10,15,19,23 - hexamethyltetracose - 2,6,10,14,18,22 - hexane), which reduces the adaptation time of microorganisms to the new substrates and ensures a faster performance of the dehydrogenation reaction of TTC and the formation of TFF. The use of higher concentrations of glucose and TTC leads to further acceleration of the target dehydrogenation reaction. Shaking the mixture after incubation homogenizes it and improves the solubility and extraction of TFF for the colorimetric determination of the optical density of the sample. The amount of formazan, which is proportional to the activity of the dehydrogenase, is assessed according to the intensity of the coloring.
Rezultatul obţinut este condiţionat de reducerea duratei de incubare a probelor datorită utilizării adaosului activant de scualenă şi majorării concentraţiei de TTC şi glucoză de 2 ori, stabilizării activităţii dehidrogenazei prin utilizarea amestecului tampon, îmbunătăţirii extragerii TFF prin agitare şi economia energiei electrice la incubarea probelor. The obtained result is conditioned by the reduction of the incubation time of the samples due to the use of the activator addition of squalene and the increase of the concentration of TTC and glucose by 2 times, the stabilization of the dehydrogenase activity by the use of the buffer mixture, the improvement of the TFF extraction by stirring and the economy of electricity when incubating the samples.
Exemplu de realizarea a invenţiei Example of the realization of the invention
Determinarea activităţii dehidrogenazei a fost efectuată în modul următor. Au fost preluate mostre din bioreactorul metanogen al gospodăriei agricole „GARMA-GRUP, SRL”, c. Fârlădeni, Hânceşti. Componenţa biomasei fermentate - 60% de borhot de la distilarea alcoolului + 40% bălegar. Dehydrogenase activity was determined as follows. Samples were taken from the methanogenic bioreactor of the farm "GARMA-GRUP, SRL", c. Fârlădeni, Hânceşti. The composition of the fermented biomass - 60% borhot from alcohol distillation + 40% dung.
Proba biomasei fermentate, cu volumul de 1 ml, a fost plasată într-o eprubetă, s-au adăugat 2 ml de soluţie tampon fosfat cu pH=7,2, 1 ml de soluţie 2% TTC, 1 ml 0,2 M de soluţie de glucoză şi scualenă (5,0…5,5)10-4% în raport cu volumul total de amestec (5 ml) incubat, care este scuturat atent. The fermented biomass sample, with a volume of 1 ml, was placed in a test tube, 2 ml of phosphate buffer solution with pH=7.2, 1 ml of 2% TTC solution, 1 ml of 0.2 M of solution of glucose and squalene (5.0...5.5) 10-4% in relation to the total volume of the incubated mixture (5 ml), which is carefully shaken.
Apoi amestecul a fost vacuumat la un vid de 10…12 mm Hg şi incubat la temperatura de 33±1°C timp de o oră în termostat. Drept control au servit materia primă (biomasa) fermentată sterilizată şi reactivii. Then the mixture was vacuumed to a vacuum of 10...12 mm Hg and incubated at a temperature of 33±1°C for one hour in a thermostat. The sterilized fermented raw material (biomass) and reagents served as control.
După finalizarea incubării în eprubetă s-au adăugat porţionat câte 10 ml de alcool etilic 96%, apoi a fost supusă agitării prin scuturare timp de 5 min. Conţinutul eprubetelor a fost apoi filtrat, iar soluţia de TFF a fost analizată la fotocolorimetru, folosind chiuvete cu lăţimea de 5 mm şi un filtru de lumină cu lungimea de undă de 540 nm. Cantitatea de trifenilformazan (în mg) a fost determinată cu ajutorul curbei standard. Pentru alcătuirea curbei de calibrare a fost preparată soluţia standard de formazan în alcool etilic (0,1 mg la 1 ml). Calculele s-au efectuat conform formulei. After completing the incubation in the test tube, 10 ml of 96% ethyl alcohol were added in portions, then it was subjected to agitation by shaking for 5 min. The contents of the test tubes were then filtered, and the TFF solution was analyzed in a photocolorimeter, using 5 mm wide sinks and a light filter with a wavelength of 540 nm. The amount of triphenylformazan (in mg) was determined using the standard curve. To create the calibration curve, the standard solution of formazan in ethyl alcohol (0.1 mg per 1 ml) was prepared. The calculations were performed according to the formula.
Exemplu de calcul conform procedeului propus: Example of calculation according to the proposed procedure:
AD = * K * 1,5*10, AD = * K * 1.5*10,
unde AD - activitatea dehidrogenazei, mg TFF/10 ml/1 oră; where AD - dehydrogenase activity, mg TFF/10 ml/1 hour;
D probă - valoarea absolută a densităţii optice a probei analizate, media a 3 măsurări; D sample - the absolute value of the optical density of the analyzed sample, the average of 3 measurements;
D probă sterilă - valoarea absolută a densităţii optice a probei sterile; D sterile sample - the absolute value of the optical density of the sterile sample;
D control - valoarea absolută a densităţii optice a reactivilor fără probă; D control - the absolute value of the optical density of reagents without sample;
t - timpul incubării, ore; t - incubation time, hours;
K - coeficientul de corecţie a densităţii optice conform curbei de calibrare, mg TFF/ml; K - optical density correction coefficient according to the calibration curve, mg TFF/ml;
1,5 - raportul dintre amestecul pentru extragere în probă (15 ml) şi în proba pentru calibrare (10 ml); 1.5 - the ratio between the mixture for extraction in the sample (15 ml) and in the sample for calibration (10 ml);
10 - recalcularea/corecţia pentru 10 ml de biomasă analizată. 10 - recalculation/correction for 10 ml of analyzed biomass.
D probă = 0,26; 0,27; 0,29 D sample = 0.26; 0.27; 0.29
D probă sterilă = 0 D sterile sample = 0
D control = 0 D control = 0
t = 0,5 t = 0.5
K = 0,641152899 K = 0.641152899
AD1 = * 0,641152899* 1,5*10 ≈ 5,00 mg TFF/10 ml/1 oră AD1 = * 0.641152899* 1.5*10 ≈ 5.00 mg TFF/10 ml/1 hour
AD2 = * 0,641152899* 1,5*10 ≈ 5,19 mg TFF/10 ml/1 oră AD2 = * 0.641152899* 1.5*10 ≈ 5.19 mg TFF/10 ml/1 hour
AD3 = * 0,641152899* 1,5*10 ≈ 5,58 mg TFF/10 ml/1oră AD3 = * 0.641152899* 1.5*10 ≈ 5.58 mg TFF/10 ml/1 hour
AD medie = (AD1 + AD2 + AD3)/3 = (5,00+5,19+5,58)/3 = 5,26 mg TFF/10ml/1ore Average AD = (AD1 + AD2 + AD3)/3 = (5.00+5.19+5.58)/3 = 5.26 mg TFF/10ml/1hour
Exemplu de calcul conform celei mai apropiate soluţii Example of calculation according to the closest solution
Calculele s-au efectuat conform formulei: The calculations were carried out according to the formula:
AD = * K * 1,3*10, AD = * K * 1.3*10,
unde: AD - activitatea dehidrogenazei, mg TFF/10 ml/1oră; where: AD - dehydrogenase activity, mg TFF/10 ml/1 hour;
D probă - valoarea absolută a densităţii optice a probei analizate, media a 3 măsurări; D sample - the absolute value of the optical density of the analyzed sample, the average of 3 measurements;
D probă sterilă - valoarea absolută a densităţii optice a probei sterile; D sterile sample - the absolute value of the optical density of the sterile sample;
D control - valoarea absolută a densităţii optice a reactivilor fără probă; D control - the absolute value of the optical density of reagents without sample;
t - timpul incubării, ore; t - incubation time, hours;
K - coeficent de corecţie a densităţii optice conform curbei de calibrare, mg TFF/ml; K - optical density correction coefficient according to the calibration curve, mg TFF/ml;
1,3 - raportul dintre amestecul pentru extragere în probă (13 ml) şi în proba pentru calibrare (10 ml); 1.3 - the ratio between the mixture for extraction in the sample (13 ml) and in the sample for calibration (10 ml);
10 - recalcularea/corecţia pentru 10 ml de biomasă analizată. 10 - recalculation/correction for 10 ml of analyzed biomass.
D probă = 0,59; 0,56; 0,59 D sample = 0.59; 0.56; 0.59
D probă sterilă = 0 D sterile sample = 0
D control = 0 D control = 0
t = 1,0 t = 1.0
K = 0,641152899 K = 0.641152899
AD1 = * 0,641152899 * 1,3*10 ≈ 4,92 mg TFF/10 ml/1oră AD1 = * 0.641152899 * 1.3*10 ≈ 4.92 mg TFF/10 ml/1 hour
AD2 = * 0,641152899 * 1,3*10 ≈ 4,67 mg TFF/10 ml/1oră AD2 = * 0.641152899 * 1.3*10 ≈ 4.67 mg TFF/10 ml/1 hour
AD3 = * 0,641152899 * 1,3*10 ≈ 4,92 mg TFF/10 ml/1oră AD3 = * 0.641152899 * 1.3*10 ≈ 4.92 mg TFF/10 ml/1 hour
AD medie = (AD1 + AD2 + AD3)/3 = (4,92+4,67+4,92)/3 = 4,84 mg TFF/10 ml/1oră Mean AD = (AD1 + AD2 + AD3)/3 = (4.92+4.67+4.92)/3 = 4.84 mg TFF/10 ml/1 hour
Rezultatele analizelor activităţii dehidrogenazei din biomasa fermentată sunt prezentate în tabel. The results of the analyzes of the dehydrogenase activity from the fermented biomass are presented in the table.
Tabel Table
Condiţii Date cantitative Procedeul conform invenţiei Conform celei mai apropiate soluţii Biomasa fermentată (60% borhot de la distilarea alcoolului + 40% bălegar) 1 ml 1 ml clorură de 2,3,5- trifeniltetrazoliu (TTC), 1% 1 ml 1 ml Concentraţia de glucoză 0,1 М - 1 ml 0,2 М 1 ml - СаСО3 - 10 mg Soluţie tampon de fosfat, pH=7,2 2 ml - Alcool etilic, 96% 10 ml 10 ml Scualenă (2,6,10,15,19,23-hexametiltetracoza -2,6,10,14,18,22-hexaen) 0,025 mg - Timpul de incubare, min 30 55 Activitatea dehidrogenazei, mg TFF(10 ml) 5,26 4,84 Conditions Quantitative data Process according to the invention According to the closest solution Fermented biomass (60% borth from alcohol distillation + 40% dung) 1 ml 1 ml 2,3,5-triphenyltetrazolium chloride (TTC), 1% 1 ml 1 ml Concentration of glucose 0.1 М - 1 ml 0.2 М 1 ml - СаСО3 - 10 mg Phosphate buffer solution, pH=7.2 2 ml - Ethyl alcohol, 96% 10 ml 10 ml Squalene (2,6,10, 15,19,23-hexamethyltetracose -2,6,10,14,18,22-hexaene) 0.025 mg - Incubation time, min 30 55 Dehydrogenase activity, mg TFF(10 ml) 5.26 4.84
Rezultatele obţinute demonstrează că durata incubării probelor analizate s-a redus de 2 ori în comparaţie cu condiţiile analizei activităţii dehidrogenazei conform procedeului cunoscut, ceea ce este condiţionat de utilizarea adaosului biologic activ de scualenă, de majorarea concentraţiilor de TTC şi glucoză, precum şi de utilizarea soluţiei tampon, ceea ce stabilizează activitatea dehidrogenazei, iar agitarea suplimentară a amestecului după incubare majorează precizia analizei datorită îmbunătăţirii solubilităţii şi a extracţiei TFF în soluţia hidroalcoolică a amestecului analizat. De rând cu aceasta, accelerarea esenţială a procesului de efectuare a analizei asigură reducerea volumului de muncă, iar concomitent se reduce consumul de energie datorită reducerii duratei de incubare a probelor în termostat. The obtained results demonstrate that the duration of the incubation of the analyzed samples was reduced by 2 times compared to the conditions for the analysis of the dehydrogenase activity according to the known procedure, which is conditioned by the use of the biologically active addition of squalene, by increasing the concentrations of TTC and glucose, as well as by the use of the buffer solution , which stabilizes the dehydrogenase activity, and the additional stirring of the mixture after incubation increases the accuracy of the analysis due to the improvement of the solubility and extraction of TFF in the hydroalcoholic solution of the analyzed mixture. Along with this, the essential acceleration of the process of carrying out the analysis ensures the reduction of the workload, and at the same time the energy consumption is reduced due to the reduction of the incubation time of the samples in the thermostat.
Nu este mai puţin importantă operativitatea tehnologică a rezultatelor analizei care este mai înaltă în comparaţie cu condiţiile de efectuare a lor în conformitate cu cea mai apropiată soluţie - procedeul cunoscut, ceea ce permite o dirijare operativă a procesului de obţinere a biogazului, de exemplu, prim introducerea adaosurilor stimulante pentru intensificarea producţiei de biogaz din biomasă şi majorarea maxim posibilă a conţinutului de biometan. No less important is the technological operativeness of the analysis results, which is higher in comparison with the conditions of their performance in accordance with the closest solution - the known process, which allows an operative management of the process of obtaining biogas, for example, first the introduction of stimulating additives for the intensification of biogas production from biomass and the maximum possible increase of the biomethane content.
1. Звягинцев Д. Г. Методы почвеной микробиологии и биологии. Москва, МГУ, 1991, р. 245-246, http://padaread.com/?book=51406&pg=251 1. Zvyagintsev D. G. Methods of soil microbiology and biology. Moscow, MGU, 1991, р. 245-246, http://padaread.com/?book=51406&pg=251
2. Инструкция по лабораторному контролю очистных сооружений на животноводческих комплексах. Часть 3. Определение биогенных веществ. Методические рекомендации по определению дегидрогеназной активности ила при технологическом контроле работы аэротенков. Москва, Колос, 1984, р. 22-24 2. Instruction on laboratory control of sewage treatment plants in animal breeding complexes. Part 3. Determination of biogenic substances. Methodical recommendations for the determination of dehydrogenase activity and the technological control of the operation of air tanks. Москва, Колос, 1984, р. 22-24
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| US4622296A (en) * | 1983-12-28 | 1986-11-11 | Wako Pure Chemical Industries, Ltd. | Process for measuring activity of dehydrogenase employing a reaction stopper |
| RU2387996C1 (en) * | 2008-09-22 | 2010-04-27 | Общество с ограниченной ответственностью "Научно-исследовательский институт природных газов и газовых технологий - Газпром ВНИИГАЗ" (ООО "ВНИИГАЗ") | Method of monitoring cleaning of soil contaminated with hydrocarbons and neutralisation of hydrocarbon sludge through analysis of dehydrogenase activity |
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| CN103525896A (en) * | 2013-09-27 | 2014-01-22 | 南京工业大学 | High-activity yeast cell quantitative screening method based on TTC staining method |
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| US4622296A (en) * | 1983-12-28 | 1986-11-11 | Wako Pure Chemical Industries, Ltd. | Process for measuring activity of dehydrogenase employing a reaction stopper |
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| RU2476598C2 (en) * | 2011-04-27 | 2013-02-27 | Федеральное государственное образовательное учреждение высшего профессионального образования "Северный (Арктический) федеральный университет" (С(А)ФУ) | Method of quantitative determination of dehydrogenase activity of microorganisms |
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