INT-脱氢酶

Ecotoxicology and Environmental Safety 53 (2002) 416–421 INT–dehydrogenase activity test for assessing anaerobic biodegradability of organic compounds Yang Hongwei,a,* Jiang Zhanpeng,a Shi Shaoqi,a and W.Z. Tangb aDepartment of Environmental Science and Engineering, Tsinghua University, Beijing 100084, China bDepartment of Civil and Environmental Engineering, Florida International University, Miami, FL 33199, USA Received 17 September 2000; accepted 13 March 2002 Abstract This study assessed anaerobic biodegradability of organic compounds from microorganism activity. Dehydrogenase activity can be a good parameter characterizing the microorganism activity. A modified method of 2-(p-iodophenyl-3-(p-nitrophenyl)-5-pheny tetrazolium chloride-dehydrogenase activity determination was proposed in anaerobic biodegradability assessment. Cubic spline curves were adopted to link the data points. This curve was integrated twice to calculate areas. The microorganism activity index in anaerobic biodegradability assessment was calculated by standardizing the integral. According to the results of the activity index, 14 kinds of organic compounds were classified into readily, partially, and poorly biodegradable under anaerobic conditions, respectively. As a result, some conclusions for anaerobic biodegradability of organic compounds were reached, based on the activity index value. r 2002 Elsevier Science (USA). All rights reserved. Keywords: Anaerobic biodegradability; Dehydrogenase activity; Microorganism activity; Cubic spline; Activity index (AI) 1. Introduction Biodegradability of organic compound can be classi- fied into aerobic and anaerobic biodegradability. It is important to assess the biodegradability of organic compounds to determine whether they are persistent in the environment. Many studies have been carried out to assess biodegradability of organic compounds. During aerobic biodegradation of an organic compound, oxy- gen uptake rate (OUR) (ISO 9408, 1991), CO2 produc- tion (Wenning and Zhanpeng, 1995), organic compound degradation (ISO 7827, 1994), and microorganism activity (Zhanpeng et al., 2000) are the four factors that are used to assess aerobic biodegradability. During anaerobic biodegradation of an organic compound, gas production (ISO 11734, 1995), organic compound degradation (Boyd and Shelton, 1984), and microorgan- ism activity are the three factors that are used to assess anaerobic biodegradability. Many methods of determin- ing anaerobic biodegradability only assess either gas production or organic compound degradation. Microorganism activity can change during biodegra- dation of organic compounds under anaerobic condi- tions. However, such a minor change in microorganism activity is not easily detected when the biomass dose not increase significantly. Standard methods, using volatile suspended solid (VSS) determination and microorgan- ism count, cannot detect such changes during anaerobic biodegradation. However, special biomolecules in mi- croorganisms can be detected as indicators of micro- organism activity. The special biomolecules are present only when the microorganism is alive, and the number of these special biomolecules will change if micro- organism activity changes. In general, special enzymes (Nybroe et al., 1992; Le Bihan and Lessard, 1998; Goel et al., 1998), such as coenzyme F420; hydrogenase, dehydrogenase (DHA), and adenosine triphosphate (ATP), may serve as indicators. Past studies on microorganism activity focused on monitoring the property of sludge during wastewater treatment to control the treatment process. Yi and Jicui (1990) *Corresponding author. Tel.: 8610-62772987; fax: 8610-62785687. E-mail address: yang98@mails.tsinghua.edu.cn (Y. Hongwei). 0147-6513/02/$ - see front matter r 2002 Elsevier Science (USA). All rights reserved. PII: S 0 1 4 7 - 6 5 1 3 ( 0 2 ) 0 0 0 0 2 - 7 studied the activity of coenzyme F420 in aerobic sludge, but the results were not consistent with the results obtained from other parameters, such as OUR. Zabranska et al. (1984) studied hydrogenase activity in aerobic sludge. Although the results indicated that this enzyme activity could characterize sludge activity, its time-consuming and complicated determination pro- cesses led to the conclusion that it was not a good parameter to monitor sludge activity. At present, only DHA and ATP have been used successfully to monitor aerobic or anaerobic sludge activity because the methods of determining them are easy and relatively quick. Chung and Neethling (1988, 1989) measured the concentration of DHA and ATP in digester sludge successfully. They also monitored the changes in DHA and ATP concentration in anaerobic sludge when there was a shock loading to the digester. These results indicated that DHA and ATP could be good candidates to serve as indicators of anaerobic microorganism activity in anaerobic degradation of organic com- pounds. In the ATP test, the storage of used chemicals is stricter than that for the DHA test and the measurement process is more complicated than that for DHA. Therefore, DHA concentration was used to indicate microorganism activity in this study. The method has been reported by Kim et al. (1994). The biodegradation of an organic compound pro- ceeds through a series of oxidation reactions involving loss of electrons or removal of hydrogen atoms from organic compounds. The process of removal of hydro- gen atoms from an organic compound is called dehydrogenation. The enzymes, which catalyze dehy- drogenation reactions, are called dehydrogenases. If the number of dehydrogenases in the biodegradation can be measured, microorganism activity can be determined. DHA is measured generally by adding a tetrazolium salt, such as triphenylteltrazolium chloride (TTC) or 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT), to a biological system. The salt is used as a terminal hydrogen acceptor in the bioreactions. The tetrazolium salt is a soluble and colorless or weakly colored chemical. After reduction by addition of two hydrogen atoms, the salt changes to a red insoluble triphenyl formazan (TF) crystal, which can dissolve in organic solvent. The following bioreactions occur: RH2 - DHA Rþ 2H; 2Hþ Tetrazolium salts-HClþ Triphenylformazan: The red TF crystals can be extracted from bacteria cells using an organic solvent. The concentration of the TF solution can be determined by measuring the solution absorbance using a spectrophotometer at 490 nm: This method (Lopez et al., 1986; Chung and Neethling, 1989) of measuring DHA is quite easy to perform and is very sensitive. In the TTC–DHA measurement, dissolved oxygen in the sample will impact the results, and variations in TTC concentration can lead to an unsteady results. In the INT–DHA measurement, dissolved oxygen in sample has little influence on the results, and an INT concentration between 0.5 and 1:0 mg=g VVS can obtain steady results if the sludge activity is steady. Therefore, the INT–DHA measure was used to assess anaerobic biodegradability of organic compounds in this study. 2. Materials and methods 2.1. Experimental method A static experiment was used in this study. The inoculum anaerobic sludge was taken from taken from the digester tanks in Gao Bei Dian wastewater treatment plant in Beijing. Before inoculum, the anaerobic sludge was cultured at 351C for about 1 week in order to reduce the organic substrate in the sludge. Then 50 mL anaerobic sludge was poured into a 600-mL serum bottle after centrifugation at 1000g: The test organic compound, the concentration of which was 100 mg=L (as TOC), was added to the serum bottle. The inorganic test medium, the ingredients of which are listed in Table 1, was added to the serum bottle until the total volume reached 500 mL: The solution pH value was then adjusted to 7:070:2 by adding sodium bicarbonate. Before the bottle was sealed, nitrogen was sparged into the bottle for about 5 min in order to remove the residual oxygen. The experimental period was 42 days, during which the culture temperature was maintained at 35711C: INT–DHA was measured by sampling from the serum bottle every several days. Simultaneously, a control experiment without adding any organic com- pounds was performed. 2.2. DHA measurement The INT reagent was from Sigma Chemical Co. (St. Louis, MO). A 2 g=L solution of INT was prepared and Table 1 Ingredients of inorganic test medium (ISO 11734, 1995) Compound Concentration=g � L�1 Na2HPO4 � 12H2O 1.12 KH2PO4 0.27 NH4Cl 0.53 CaCl2 � 2H2O 0.075 MgCl2 � 6H2O 0.1 FeCl2 � 2H2O 0.02 Na2S � 9H2O 0.1 Trace elements 0.001 Y. Hongwei et al. / Ecotoxicology and Environmental Safety 53 (2002) 416–421 417 stored below 41C: Because the experiment was static, the volume of the sample had to great enough not to result in too small a ratio of residual volume/total volume. Therefore, a modified method for INT–DHA determi- nation was proposed as follows, based on the method proposed by Chung and Neethling (1989): * The mixture in the serum bottle was mixed with a magnetic stirrer for about 2 min in order to mix the solution and anaerobic sludge sufficiently. Then 0:5 mL was removed a placed in a 10-mL tube with a cap. * One-tenth milliliter prepared INT solution was added to the tube. After the tube was tightly capped, it was incubated in a water bath at 351C for 1 h: During the incubation, the tube was inverted every 15 min to resuspended the solids. * After incubation, 0:2 mL of 37% formalin was added to the tube to stop the reaction. * The mixture was centrifuged at 1200g for 15 min and the supernatant was discarded. * The TF crystal was extracted by adding 5 mL ethyl acetate for 30 min in the dark. The tube was inverted every 15 min: * The mixture was centrifuged at 600g for 5 min after the TF was extracted. The supernatant was sepa- rated to measure the absorbance in a spectro- photometer at 490 nm: 3. Results and discussion This study measured the INT–DHA during the anaerobic biodegradation of 14 organic compounds. These compounds can be classified into two categories, six were fatty acid and the others were aromatic compounds. The results of measurements are found in Figs. 1 and 2. Different organic compounds have different INT–DHA curves. In the current experiment there were four types of curves. Fig. 3 illustrates these. Some organic compounds can be biodegraded by anaerobic microorganisms immediately. After the che- mical was added to the sludge, the INT–DHA of the sludge increased and reached the maximum value quickly. Curve A characterizes the biodegradation process of such organic compounds, which can be considered readily biodegradable. In this study, all of the fatty acids fall in this class. Some organic compounds could not be biodegraded at the beginning of the experiments, but could be utilized by the anaerobic microorganisms after a period of adaptation. Curve B represents the activity of these compounds. After the period of adaptation, the INT–DHA increased quickly and reached maximum value. The INT–DHA curves of phenyl-acetic acid and 2-methyl-pheno belong to curve B. Such organic compounds can be considered readily or partially biodegradable. Some organic compounds could be biodegraded to certain intermediate products. Such intermediate pro- ducts could be biodegraded to end products by anaerobic microorganisms after a period of adaptation. The INT–DHA curve increased to a peak valve and then decreased. After the period of adaptation, the curve increased again to a higher peak value. Curve C characterizes such changes during biodegradation of Fig. 1. INT–DHA in anaerobic sludge during fatty acid biodegrada- tion. (—~—) Control; (—’—) formic acid; (—m—) acetic acid; (— �—) propionic acid; (—*—) butyric acid; (—�—) valeric acid; (—j—) hexylic acid. Fig. 2. INT–DHA in anaerobic sludge during aromatic compounds biodegradation. (—~—) Control; (—’—) phthalic acid; (—m—) 4- methyl-benzoic acid; (—�—) phenyl-acetic acid; (—*—) 2-methyl- phenol; (—�—) 3-methyl-phenol; (—j—) benzene-1,2-diol; (–) 4- amino-phenol; (—) 4-nitro-phenol. Fig. 3. INT–DHA in anaerobic sludge of organic compounds biodegradation. Y. Hongwei et al. / Ecotoxicology and Environmental Safety 53 (2002) 416–421418 organic compounds. 3-Methyl-phenol belongs to curve C. Such compounds can be considered readily or partially biodegradable. Some organic compounds could not be biodegraded until the end of the experiment. Furthermore, during the incubation, anaerobic microorganism activity could not reach a peak value. INT–DHA decreased at first because the microorganisms were inhibited by the chemicals. At the end of the incubation, activity began to increase slowly. Curve D characterizes such changes in INT–DHA during biodegradation of organic com- pounds. 4-Nitro-phenol belongs to curve D. Such organic compounds can be considered poorly biode- gradable. The qualitative assessment method just described for classification of biodegradability of organic compounds under anaerobic conditions based on their INT–DHA curves does not accurately classify the anaerobic biodegradability of organic compounds. Therefore, a new quantitative assessment method was proposed. From the shapes of INT–DHA curves, it can be concluded that the peak value of the curve and the time of peak appearance are the two key parameters in biodegradability assessment. However, only two such parameters cannot reflect all the information given by the INT–DHA curves. There is more useful information that can impact anaerobic biodegradability assessment of organic compounds. For examples, curve C has two peaks (Fig. 3); therefore the value of two parameters could not be determined uniquely. If two INT–DHA curves have the same peak value and the time of peak appearance, but had different peak widths, the results of the quantitative assessment must be different. A more rational and more accurate assessment index should be established based on the INT–DHA curves. INT–DHA data points are difficult to regress with polynomials or single functions because of the abnormal shapes of the curves. In order to obtain a smooth curve to link the data points, cubic spline lines are used. The area between the curve and abscissa can contain the information of peak high and peak width, and the greater the area the more easily the organic compound is biodegraded. However, such a simple assessment index cannot include the information of the time of peak appearance. For example, if two curves have the same area, but the times of peak appearance are different, such as curves A and B in Fig. 3, it is obvious that the organic compounds with curve A are more easily biodegraded under anaerobic conditions than the organic compounds with curve B. In order to consider the time of peak appearance in the assessment index, the integrated curve of the cubic spline lines found in Fig. 4 should be analyzed. The area between the integrated curve and the abscissa can contain information about the time of peak appearance. The time of peak appearance of curve A in Fig. 3 is earlier than that of curve B. Its integrated curve in Fig. 4 increases earlier and more quickly, and the area of integrated curve is greater, so its anaerobic biodegradability is easier. Therefore, the area is calculated by integrating the cubic spline lines twice. Such an area can contain all the information in the INT–DHA curve that impacts the anaerobic biodegradability assessment. The net area of an organic compound is calculated by subtracting that of the control in order to reduce errors caused by the different conditions between experiments, especially the different properties of anaerobic sludge. The activity index (AI) is calculated by standardizing the integral. An example of the standardization process is as follows: an AI for benzene-1,2-diol of 1 is hypothesized, and then AI values for other compounds is the ratio of their net areas/the net areas of benzene-1,2-diol. In different experiments, benzene-1,2-diol was tested to reduce the errors caused by different experimental conditions. The detailed process of AI calculation can be described using the equations A ¼ f ðxÞ; F ðxÞ ¼ Z f ðxÞ dx; S ¼ Xn�1 i¼1 Z xjþ1 xi F ðxÞi dx; AI ¼ S � Scontrol Sbenzene-1;2-diol � Scontrol ; where A is the absorbance of TF solution at 490 nm using spectrophotometer, x is sampling time, f ðxÞ is a series cubic spline functions to link the data points, F ðxÞ is the integrated of cubic spline functions; S is the area between F ðxÞ and the abscissa, Scontrol is the area of the control test, Sbenzene-1;2-diol is the area of the benzene-1, 2-biol test, and AI is the standardized activity index of the biodegradability of an organic compound under anaerobic conditions. All calculations discussed were performed using MATLAB5.3. During the data processing, the absorbance of the TF solution was used directly and the concentrations of Fig. 4. Intergrated curve of curves A and B from Fig. 3. Y. Hongwei et al. / Ecotoxicology and Environmental Safety 53 (2002) 416–421 419 INT–DHA in the anaerobic sludge were not calculated. After the absorbance of TF was calculated, the concentrations of INT–DHA were compared. From the process of TF formations, it is obvious that the amount TF formed is directly proportional to the INT–DHA in anaerobic sludge, and therefore the concentration of the TF solution is proportional to the concentration of INT–DHA. Furthermore, the absorbance of TF is linear to the concentration of the TF solution and can be described as CTF ¼ kATF þ b; where ATF is the absorbance of TF, CTF is the concentration of TF, and k and b are constants. Therefore, CINT2DHA ¼ KATF þ b; where CINT2DHA is the concentration of INT–DHA, and K and b are constants. In the following derivation, the subscript c is for the INT–DHA concentration, and the subscript A is for the absorbance of the TF solution: f ðxÞc ¼Kf ðxÞA þ b -F ðxÞc ¼ KF ðxÞA þ bx -Sc ¼ KSA þ 0:5b Xn�1 i¼1 ðx2iþ1 � x 2 i Þ -AIc ¼ KðSA � ScontrolÞ þ Xn�1 i¼1 ðð0:5bðx2iþ1 � x 2 i ÞÞ � ð0:5bðx 2 iþ1 � x 2 i ÞÞcontrolÞ= KðSbenzene-1;2-diol � ScontrolÞ þ Xn�1 i ðð0:5bðx2iþ1 � x 2 i ÞÞbenzene-1;2-diol � ð0:5bðx2iþ1 � x 2 i ÞÞcontrolÞÞ: The xi of data points must be the same for different INT–DHA curves because of the same sampling time, so the value of S in the AI formula must be zero. It can be concluded that AIC is equal to AIA: That is to say, the same value of AI can be obtained whether the data for the absorbance of the TF solution or those for the concentration of INT–DHA were adopted. Table 2 lists the results of the calculations and the qualitative assessments. From the table, the following conclusions can be reached: * For short-chain fatty acids, the longer the chain, the greater is the AI value, and the easier is anaerobic biodegradability. * For aromatic compounds, the compounds substi- tuted by nitro are less easily biodegraded; those with hydroxyl substitutions are more easily biode- graded. For example, anaerobic biodegradability of 4-nitro-phenol occurs less easily than that of 4-amino-phenol, and anaerobic biodegradability of 2-methyl-phenol is more difficult than of benzene- 1,2-diol. * For phenol compounds, the compounds substituted on the m- sides are biodegraded more readily; for example, 3-methyl-phenol is biodegraded more readily than 2-methyl-phenol. * For isomeric compounds of aromatic acids, com- pounds with single substitutions on the benzene cycle are more readily biodegraded. For example, phenyl-acetic acid biodegrades more readily than 4-metyl-benzoic acid. The current results agree with those of Xingzhi et al. (2000), in concluding that the AI of or- ganic compound biodegradability under anaerobic conditions based on microorganism activity is a viable method. Table 2 Results of AI calculation of organic compounds and qualitative assessment No. Organi