Environ. Scl. Technol. 1988.22, 1160-1165
seasonal changes on the accumulation of HzOZin a natural water. In the environment, the profiles of Hz02accumulation rate will not necessarily appear as illustrated in Figures 1 and 2. The calculations that resulted in these figures have not taken into account turbulent mixing. Mixing in natural waters is generally considerable when the surface wind is more than a few knots or in the case of flowing streams. Mixing will both dilute H20zdown into the water column and bring water not previously exposed to sunlight to the surface (26). Furthermore, non-light-induced decay rates are likely to vary in different waters depending on such factors as suspended particulate loading, water quality, and biological activity. Other than the recent work on algae (27))these factors have not as yet been studied, and a quantitative knowledge of these decay processes and rates is necessary before diel concentrations of HzOz can be constructed. Conclusions
Hydrogen peroxide is formed photochemically in natural waters containing light-absorbing organic substances. The accumulation rate of H202is related to the concentration of these natural substances and appears to be ubiquitous in its occurrence in natural waters. From the experimentally derived quantum yields, model calculations of Hz02 accumulation rate agree with experimentally determined rates for surface waters. Registry No. HzOz, 7722-84-1. Literature Cited Zafiriou, 0. C.; Joussot-Dubien, J.; Zepp, R. G.; Zika, R. G. Environ. Sci. Technol. 1984, 18, 358A-71A. Moffett, J. W.; Zika, R. G. Mar. Chem. 1983, 13, 239-51. Miles, C. J.; Brezonik, P. L. Environ. Sci. Technol. 1980, 15, 1089-95. Collienne, R. A. Limnol. Oceanogr. 1983,28,83-100. Sinel'nikov, V. E. Gidrobiol. Zh. 1971,7,115-9 (in Russian). Sinel'nikov, V. E.; Demina, V. I. Gidrokim. Mater. 1974, 60, 30-40 (in Russian). Sinel'nikov, V. E.; Liberman, A. S.Tr.-Inst. Biol. Vnutr. Vod Adad. Nauk SSSR 1974, No. 29,27-40 (in Russian). Draper, W. M.; Crosby, D. G. Arch. Environ. Contam. Toxicol. 1983, 12, 121-6. Cooper, W. J.; Zika, R. G. Science (Washington,D.C.) 1983, 220,711-2. Helz, G. R.; Kieber, R. J. Water Chlorination: Chem. Environ.Impact Health Eff. Proc. Conf.1985,5th, 1033-40. Van Baalen, C.; Marler, J. E. Nature (London) 1986,211, 951.
Zika, R. G. Ph.D. Thesis, Dalhousie University, Halifax, NS, 1978, 346 pp. Zika, R. G. In Marine Organic Chemistry;Duursma, E. K., Dawson, R., Eds.; Elsevier: Amsterdam, 1981; pp 299-325. Zika, R. G. EOS, Trans. Am. Geophys. Union 1980, 61, 1010. Zika, R. G.; Saltzman, E. S.; Cooper, W. J. Mar. Chem. 1985, 17, 265-75. Zika, R. G.; Moffett, J. W.; Petasne, R. G.; Cooper, W. J.; Saltzman, E. S. Geochim. Cosmochim. Acta 1985, 49, 1173-85. Baxter, R. M.; Carey, J. H. Nature (London) 1983, 306, 575-6. Draper, W. M.; Crosby, D. C. J. Agric. Food Chem. 1983, 31 , 734-7. Petasne, R. G.; Zika, R. G. Nature (London) 1987, 325, 516-8. Oliver, B. G.; Cosgrove, E. G.; Carey, J. H. Environ. Sci. Technol. 1979, 13; 1075-7. Zepp, R. G.; Wolfe, N. L.; Baughman, G. L.; Hollis, R. C. Nature (London) 1977,267, 421-3. Draper, W. M.; Crosby, D. G. J. Agric. Food Chem. 1981, 29, 699-702. Hoffman, M.; Edwards, J. 0. Inorg. Chem. 1977,16,3333-8. Skurlatov, Y. 1.; Zepp, R. G.; Raugman, G. L. J.Agric. Food Chem. 1983,31,1065-71. Zepp, R. G.; Schlotzhauer, P. F.; Simmons, M. S.; Miller, G. C.; Baughman, G. L.; Wolfe, N. L. Fresenius' 2. Anal. Chem. 1984,319, 119-25. Plane, J. M. C.; Zika, R. G.; Zepp, R. G.; Burns, L. A. In Photochemistry of Environmental Aquatic Systems; Zika, R. G., Cooper, W. J., Eds.; ACS Symposium Series 327; American Chemical Society: Washington, DC, 1987; pp 250-67. Zepp, R. G.; Skurlatov, Y. L.; Pierce, J. T. In Photochemistry of Environmental Aquatic Systems; Zika, R. G., Cooper, W. J., Eds.; ACS Symposium Series 327; American Chemical Society: Washington, DC, 1987; pp 215-24. Holm, T.; George, G. K.; Barcelona, M. J. Anal. Chem. 1987, 59, 582-6. Zepp, R. G.; Cline, D. M. Enuiron. Sci. Technol. 1977,11, 359-66. Kieber, R. J.; Helz, G. R. Anal. Chem. 1986,58, 2311-5. Hatchard, G. C.; Parker, C. A. Proc. R. SOC.London, A 1956, 235, 518-36. Wagner, E. E.; Adamson, A. W. J. Am. Chem. SOC.1966, 88, 394-404.
Received for review July 20,1984. Revised manuscript received October 24,1986. Accepted March 3,1988. Financial support for the research was provided by Office of Naval Research Contract NOOO14-85C-0020 and National Science Foundation Grant OCE 78-25628 and by the Drinking Water Research Center, Florida International University, Miami, FL.
Biodegradation Studies of Aniline and Nitrobenzene in Aniline Plant Waste Water by Gas Chromatography Sampatrao S. Patil and Vijay M. Shlnde"
Analytical Chemistry Laboratory, Institute of Science, Bombay 400 032, India
rn A gas chromatographic (GC) method has been developed for studying the biodegradation of aniline and/or nitrobenzene in aniline plant waste water. The effects of various parameters have been reported and critically discussed. The results are precise and afford simultaneous determinations of aniline and nitrobenzene. Introduction
Aniline and nitrobenzene are used extensively in the 1160
Environ. Sci. Technol., Vol. 22, No. 10, 1988
industries manufacturing synthetic resins, pesticides, dyestuffs, drugs, photographic chemicals, varnishes, vulcanization accelerators, and antioxidants. They are discharged in the aqueous waste which may subsequently accumulate in the environment and prove toxic to living forms. Nitrobenzene is categorized as a priority pollutant by the EPA (1). It causes cyanosis and skin and eye irritation; affects the central nervous system; and produces fatigue, headache, slight dizziness, vertigo, and vomiting (2). Its continuous exposure leads to liver damage,
0013-936X/88/0922-1160$01.50/0
0 1988 American Chemical Society
jaundice, and anemia (3). It has also been reported to inhibit cell multiplication of Pseudomonas putida at 7 mg L-' and that of Microcystis aeruginosa at 1.9 mg L-l. The LCo values for Escherichia coli, Scenedesmus, and Daphnia were evaluated at 600, 40, and 28 mg L-l, respectively. The 6-h LCsofor fish [Vairon (F)]in distilled water and hard water ranged from 20 to 24 and from 90 to IO0 mg L-l, respectively (4). Aniline causes cyanosis and depression of the central nervous system (5). It has been reported to inhibit cell multiplication of M. aeruginosa at 0.16 mg L-' and that of P. putida at 130 mg L-l. It has also been proved toxic to Scenedesmus and Daphnia a t 10 and 0.4 mg L-l, respectively ( 4 ) . In view of these environmental deleterious effects of both nitrobenzene and aniline, their biodegradability is of great importance. Most biodegradation work is based on indirect methods including BOD, COD, and TOG (6, 7). Bogatyrev (8)has studied the influence of aromatic nitrated hydrocarbons on the activated sludge process and found the partial suppression of nitrification. Pitter (9) has investigated the monosubstrate biodegradation for aniline and nitrobenzene by COD. Gomolka and Gomolka (10) have found that the nitrobenzene is biodegradable by municipal waste water. Chudoba and Pitter (7) have studied the biological purification of waste water obtained from a nitrobenzene manufacturing plant. The rate of biodegradation on COD basis was found to decrease due to the presence of dinitrophenol and dinitrocresol. Gvozdyak et al. (11)have studied the aniline biodegradation in model waste water and found that the rate of degradation was dependent on the initial aniline concentration. Suzuki and Fujii (12) observed that in the treatment of waste water containing benzoic acid, catechol, benzaldehyde, and p-cresol the biodegradation rates were nearly equal in all the cases. Deshpande et al. (13) have studied the biodegradation of rn-aminophenol manufacturing waste water containing m-nitrobenzenesulfonate, resorcinol, and rn-aminophenol. They observed that the removal of resorcinol and maminophenol was complete whereas rn-nitrobenzenesulfonate could be removed only partially. Austern et al. (14) have used extraction with freon and a gas chromatographic method for the determination of nitrobenzene added to waste water. The EPA (15) has reported a gas chromatographic method for the analysis of nitrobenzene, after its extraction with methylene chloride, from municipal and industrial waste water discharges. GC has also been used for detection and determination of other aromatic compounds like phenols, cresols, xylene, toluene, and benzene in waste waters (16, 17). In this paper we report a systematic study on the biodegradation of aniline and nitrobenzene in monosubstrate, binary substrate, and aniline plant waste water system by flame ionization gas chromatography using a Tenex column. This method has proved to be rapid; the total analysis time being less than 10 min and provides direct determinations of both nitrobenzene and aniline, Experimental Section Acclimation. The biomass used in the present investigation was obtained from a sewage treatment plant treating domestic sewage on the extended aeration principle. Acclimation experiments were carried out by batch process in glass aeration bottles (6.5-L capacity) marked as bioreactor I and bioreactor 11. Soil samples which had been exposed for more than 15 years to the aqueous waste of nitrobenzene, nitrotoluene, aniline, and m-aminophenol manufacturing units (nitrogenous waste) were selected. The samples from the discharge point (18)were collected and 50 g of homogenized soil was mixed with 200 mL of
tap water. The mixture was allowed to settle, and the supernatant (around 100 mL) was added to bioreactor I. This addition was done only once at the initiation of the experiment. The bioreactor I1 did not contain discharge point soil wash water. The biomass in the form of sludge was added to each bioreactor in the quantities which would give around 4000 mg L-l mixed liquor volatile suspended solids (MLVSS) in a final volume of 5 L. The aniline plant waste water was collected, analyzed, and found to contain 7565 mg L-l chemical oxygen demand (COD), 2010 mg L-' nitrobenzene, and 1800 mg L-l aniline. This waste water was used for the acclimation process. The waste water addition was initiated with 100 mL per day, which was increased finally to 500 mL, the increment being on the order of 100 mL. Initially the mixed liquor volume was made up with sewage and was gradually replaced with tap water during the progress of acclimatization. Salts such as ammonium sulfate (250 mg/L), potassium dihydrogen phosphate (10 mg/L), dipotassium hydrogen phosphate (26 mg/L), and disodium hydrogen phosphate, dodecahydrate (54 mg/L), were added daily as nutrients in a final volume of 5 L. Aeration was maintained at a rate sufficient to keep the solids in suspension. Both bioreactors I and I1 were operated in a semicontinuous mode using filland-draw technique; the cycle time used was 22 h. At the end of each cycle, the sludge was allowed to settle for 0.5 h, and around 3.5 L of supernatant was withdrawn. The settled sludge was washed twice with tap water and used for the next cycle. The acclimatization process was monitored by determining the soluble COD of the contents of bioreactor I and I1 collected in the beginning and at the end of the each cycle. The biomass was considered acclimated when it reduced the waste water COD content to the extent of around 90-9590. The period of acclimatization was 15 days. Monosubstrate and Binary Substrate Biodegradation. Bench-scale batch bioreactors I and I1 using acclimated activated sludge were employed to study the biodegradability of aniline, nitrobenzene, and a mixture of aniline and nitrobenzene. The biodegradation systems include monosubstrate containing either aniline or nitrobenzene in the tap water and binary substrate, which is a mixture of aniline and nitrobenzene, in the tap water. Bioreactors I and I1 contained 184 mg L-I nitrobenzene in the first experiment, 215 mg L-' aniline in the second experiment, and a mixture of 223 mg L-l aniline and 222 mg L-l nitrobenzene in the third experiment besides the required quantities of nitrogen and phosphorus as mentioned earlier. For each system a control containing substrate(s) and nutrients was maintained to determine the extent of substrate removal by air stripping. The bioreactor contents were aerated for 5-7 h, and samples (40 mL mixed liquor from each bioreactor) were withdrawn at regular intervals, filtered, and preserved for analysis. The sludges collected were kept under aerobic conditions and then returned to the respective bioreactor, after the biodegradation rate run. The aeration was continued till the end of the cycle. The sludge, after washing and reacclimatization with aniline plant waste water, was used for the next biodegradation experiments. pH, chemical oxygen demand (COD), mixed liquor suspended solids (MLSS), and mixed liquor volatile suspended solids (MLVSS) were determined by standard methods (19). Chromatographic analyses for aniline and nitrobenzene were conducted as described below. Gas Chromatography. Analyses of aniline, nitrobenzene, and a mixture of aniline and nitrobenzene were performed on a Varian 3700 gas chromatograph equipped Environ. Sci. Technol., Vol. 22, No. 10, 1988
1161
Table I. Characteristics of Waste Water from Aniline Manufacturea
I:
parameter
min
max
av
SD
PH total solids COD nitrobenzene aniline
8.1 140 4554 1520 650
10.0 190 8350 2010 2105
163 6007 1828 1230
1355 159 502
17
"Results are the average of eight sets of composite waste (each set collected on different days). All values except pH are expressed as mg Table 11. Recovery of Aniline (AN) and Nitrobenzene (NB) Spiked into Tap Water (Amounts in mg L-I)
set 0
10 TIME ( m i n i
Flgure 1. Direct analysis of aniline and nitrobenzene in tap water. Chromatogram of water sample spiked with 11 1.48 mg 1-' of aniline (B), 122.48 mg L-' of nitrobenzene (C), and 283.1 mg L-' of CHA (A). Internal standard injected onto a glass column 0.25 in. 0.d. X 2 mm i.d. X 200 cm containing Tenex GC at 180 OC.
with a dual flame ionization detector, VA-9176 recorder, and minigrator. Injector port and detector were at 250 "C, and the column was at 180 OC. Nitrogen carrier gas flow was maintained at 20 mL min-' through a glass column 0.25 in. 0.d. X 2 mm i.d. X 200 cm packed with Tenex GC, 60-80 mesh. Aqueous samples, 2-mL in size, containing 0.028% cyclohexylamine as an internal standard were injected directly into the gas chromatograph. Relative response factors were determined for both aniline and nitrobenzene and used for recovery study and to analyze the contents of bioreactors I and 11. Waste Water Biodegradation. Aniline plant waste water samples of the same volume were collected for every 2 h over a period of 24 h, and these samples were mixed to prepare the composite sample. This composite waste water was used for characterization in terms of pH, total solids, and COD as per the standard methods (19). The aniline and nitrobenzene contents were determined by gas chromatographic method as described above. For studying the waste water biodegradation, equal quantities of aniline plant waste water were added to bioreactors I and I1 and diluted with tap water. Both the reactors were found to contain 150 mg L-l of aniline and 250 mg L-l of nitrobenzene by gas chromatography. The required quantities of nitrogen and phosphorus were also supplied. The experiment was conducted for 8 h, and samples (40 mL of mixed liquor from each bioreactor) were withdrawn at regular intervals, filtered, and analyzed for aniline and nitrobenzene by the gas chromatographic method as described earlier. pH, COD, MLSS, and MLVSS were determined by standard methods (19).The sludges collected were kept under aerobic condition and then returned to the respective bioreactor, after the biodegradation rate run. Results and Discussion Characteristics of waste water from the aniline manufacturing plant are shown in Table I. A typical chromatogram obtained from 2 p L of an aqueous solution containing aniline, nitrobenzene, and cyclohexylamine (CHA) as internal standards is illustrated in Figure 1. Similarly, aniline plant waste water sample was chromatographed by direct injection and is shown in Figure 2. The retention times (in s) of CHA, aniline, and nitrobenzene are 137,308, and 485, respectively, in both spiked water and waste 1162
Environ. Sci. Technol., Voi. 22, No. 10, 1988
1
2 3 4
5 6 7
8 9
AN NB AN NB AN NB AN NB AN NB AN NB AN NB AN NB AN NB
0
amount taken
amount found
% error
13.9 15.3 27.9 30.6 41.8 45.9 55.7 61.2 111.5 122.5 223.0 244.9 320.4 355.4 534.0 592.3 747.5 829.2
13.8 15.0 27.7 31.4 41.7 46.0 58.0 63.5 111.7 117.8 222.5 252.4 324.9 360.1 527.2 570.2 778.7 841.1
-1.0
-2.0 -0.7 +2.5 -0.2 +0.2 +4.1 +3.7 +0.2 -3.8 -0.2 +3.1 +1.4 +1.3 -1.3 -3.7 +4.2 +1.4
10 T I M E (mln)
Flgure 2. Direct waste water sample analysls. Chromatogram of waste water containing aniline (B),nitrobenzene (C), and CHA (A). Internal standard column as in Figure 1.
water. Constituents in the waste water were identified and quantitative analyses were made by the internal standard method. The recoveries of aniline and nitrobenzene from standard mixtures in the tap water are given in Table 11. The observed error of the method is less than f4.2%. The statistical evaluation of the method is given in Table 111. The reproducibility of the quantitative measurement is quite good as indicated by the coefficient of variation ranging from 0.4 to 2.9% for the three sets containing different levels of nitrobenzene and aniline. This method is practicable, accurate, and rapid. In additions it does not require tedious pretreatment of samples and avoids possible losses. The disappearance of peaks of aniline and nitrobenzene from the monosubstrate, binary substrate, and waste water
B
I
L TIME
(mi")
system using bioreactors I and I1 was monitored by this gas chromatographic method. Results are shown in Tables IV-VII. In the above systems, removal of aniline and nitrobenzene is found to be complete, whereas the control test without activated sludge and with continuous aeration for 6-7 h shows negligible stripping of constituents as shown by GC analysis. The chromatograms showing the course of biodegradation of binary substrate and waste water system have been shown in Figures 3 and 4, respectively, whereas traces of aniline (Figure 3) and nitrobenzene (figure 4) still remain; however, subsequently they are completely removed if the biodegradation is continued for another hour. The removal observed is simultaneous and confirms that both aniline and nitrobenzene are biodegradable in presence of each other. The metabolic intermediate in the biodegradation process was not traceable
Flgure 3. Chromatograms showing biodegradation of a mixture of (B) aniline, (C) nitrobenzene, and (A) CHA. Table 111. Statistical Evaluation of Method for Analysis of Aniline (AN) and Nitrobenzene (NB) Spiked into Tap Water at Different Levels (Amounts in mg L-l)
set
I
I1 I11
amount taken
amount found'
27.9 30.6 106.8 118.5 320.4 355.4
27.8 31.5 107.2
AN NB AN NB AN NB
118.2
322.0 355.9
SD
coeff of variance, %
0.1 0.1 1.5 3.4 2.2 4.0
:'~Efntn
liME(min)
Flgure 4. Chromatograms showing biodegradation of aniline piant waste water system containing (B) aniline (C) nitrobenzene, and (A) CHA.
10
I
10 TIME i m i n )
10 iIME(min)
0.4 0.4 1.4 2.9 0.7 1.1
'Average of five determinations. Table IV. Biodegradation of Aniline (AN)
control aeration time, h 0 2 4 6 7
a1
AN remaining, mg L-'
bioreactor I," 1 = 0.05 g of AN (E! of MLVSSP AN COD remaining, remaining, mg L-l mg L-' I
COD remaining, mg L-'
215 493 212 490 216 492 216 494 214 492 MLSS, mg L-' MLVSS, mg L-' ultimate biodegradation rate, mg COD removal g-' MLVSS h-l
215 175 108 5 nil
493 398 245 61 47 5810 4270 17
bioreactor 11," 1 = 0.048 g of AN (E .- of MLVSS)-' AN COD remaining, remaining, mg L-' mg L-' 215 178 109 7 nil
493 405 247 68 54 6080 4500 16
= loading. Results are the average of three experiments for each bioreactor.
Table V. Biodegradation of Nitrobenzene (NB)
control
bioreactor I," 1 = 0.043 g of NB (g of MLVSS)-l NB COD remaining, remaining, mg L-' mg L-' 183 329 114 203 46 83 7 29 5790 4260 14
bioreactor 11," 1 = 0.041 g of NB (g of MLVSS)-' NB COD remaining, remaining, mg L-l mg L-' 183 329 121 215 41 72 6 37 6080 4500
NB COD remaining, remaining, aeration time, h mg L-' mg L-' 0 183 329 2 183 327 4 183 326 5 179 320 MLSS, mg L-' MLVSS, mg L-l ultimate biodegradation rate, 13 mg COD removal g-l MLVSS h-l 'I = loading. At 6-h interval, NB peaks were not observed. Results are the average of three experiments for each bioreactor.
Environ. Sci. Technol., Vol. 22, No. 10, 1988
1163
Table VI. Biodegradation of a Mixture of Aniline (AN) and Nitrobenzene (NB)
aeration time, h
AN remaining, mg L-'
0 2 4 6 7 8
223 223 225 224 222 223
control NB remaining, mg I,-'
+
COD remaining, mg L-'
bioreactor 1', 1 = 0.052 g of AN 0.052 g of NB (g of MLVSS)-' AN NB COD remaining, remaining, remaining, mg L-' mg L-' mg L-'
222 911 221 914 224 913 223 910 223 912 221 913 MLSS, mg L" MLVSS, mg L-I ultimate biodegradation rate, mg COD removal g-' MLVSS h-l
223 179 118 7 nil nil
222 162 101 32 nil nil
+
bioreactor 11," 1 = 0.049 g of AN 0.049 g of NB (g of MLVSS)-' AN NB COD remaining, remaining, remaining, mg L-' mg L-' mg L-'
911 705 455 73 65 55
223 187 134 63 7 nil
5800 4260 33
222 155 85 29 nil nil
911 708 460 198 95 59
6100 4530 26
1 = loading. Results are the average of three experiments for each bioreactor. Table VII. Biodegradation of Aniline Plant Waste Water Containing Aniline (AN) and Nitrobenzene (NB)
aeration time, h
AN remaining, mg L-'
control NB remaining, mg L-'
COD remaining, mg L-*
0 2 4 5 6
150 153 151 151 151
250 253 249 251 250
784 786 784 782 784
bioreactor I," 1 = 0.033 g of AN + 0.058 g of NB (g of MLVSS)-' AN NB COD remaining, remaining, remaining, mg L-l mg L-' mg L-' 150 88 1 nil nil
250 158 80 40 nil
MLSS, mg L-I MLVSS, mg L-' ultimate biodegradation rate, mg COD removal g-' MLVSS h-'
+
bioreactor 11,' 1 = 0.034 g of AN 0.060 g of NB (g of MLVSS)-' AN NB COD remaining, remaining, remaining, mg L-' mg L-' mg L-'
784 488 147 73 39
150 107 21 nil nil
6080 4270 33
250 172 99 64 24
784 556 227 114 94
5650 4160 28
1 = loading. At 7-h intervals, NB peak was not observed in the case of bioreactor 11. Results are the average of three experiments for each bioreactor.
Table VIII. Biodegradabilitya of Aniline (AN) and Nitrobenzene (NB)
biodegradation system
bioreactor I rate of degradation, mg of Sb (g of MLSS)-' h-l
bioreactor I1 rate of degradation, mg of S (g of MLSS)-' h-'
AN monosubstrate AN binary substrate AN waste water system NB monosubstrate NB binary substrate NB waste water system
6.0 6.2 6.1 6.1 5.5 6.9
5.1 5.1 5.7 5.8 5.3 6.7
bioreactor IC rate of degradation, 1 = g of S (g of mg of S (g of MLVSS)-l h-l MLVSS)-l 0.050 0.052 0.033 0.043 0.052 0.058
8.2 8.5 8.7 8.3 7.5 9.8
Represents the primary biodegradation rates. bSubstrate. 1 = loading. Temperature = 26-28
as no peaks other than parent peaks were detected by GC. Table VI11 shows the organic loading and removal rates for different biodegradation systems. Though the organic loadings vary from 0.033 to 0.052 and from 0.041 to 0.060 g per gram of MLVSS for aniline and nitrobenzene, respectively, the biodegradation rates are nearly equal for proposed systems. Aniline and nitrobenzene proportion is around 1:1.6 in the waste water system and 1:l in the binary substrate. >However,the proportion of these two substrates does not play any role in influencing the removal efficiency. The soil culture obtained from the waste water discharge point was found to be more efficient in bioreactor I system. This was expected because the culture had been exposed to nitrogenous waste for the last 15 years. Since waste water was used for the acclimatization of activated sludge, the removal rates are comparatively high for the waste water system. The biodegradability observed by GC method shows the primary biodegradation as given in Table VIII. The ultimate biodegradation in terms of COD has also been given 1164 Environ. Sci. Technoi., Vol. 22, No. 10, 1988
bioreactor 11' rate of 1 = g of S degradation (g of mg of S (go of MLVSS)-l MLVSS)-l h-l 0.048 0.049 0.034 0.041 0.049 0.060
7.7 6.8 7.7 7.8 7.1 9.1
OC.
in Tables IV-VII. Although the primary biodegradation of the substrates shows 100% removal, the ultimate biodegradation in terms of COD is in the range of 90-95%. The COD removal rates observed in binary and waste water systems were almost the same, whereas in the monosubstrate system it was found to be more or less half of the binary or waste water system for nearly the same substrate loadings. The substrate removal rates observed in monosubstrate, binary substrate, and waste water systems were nearly equal for both aniline and nitrobenzene. This is in conformation with the one observed by Suzuki and Fujii (12) for the biodegradation studiesof waste water containing benzoic acid, catechol, benzaldehyde, and p cresol. As time progressed the pH of the monosubstrate and binary substrate systems changed as shown in Figure 5. In all the three cases there was an initial increase in pH which decreased after 5-7 h. The maximum increase in pH correlated well with almost complete removal of substrates. In the case of aniline, the increase may be due to
I
( 5 ) Change in pH due t o biochemical reaction has no effect on the degradation process. (6) The discharge point soil culture is more efficient than the culture developed only with domestic sewage and waste water. (7) Aniline and nitrobenzene in waste water are biodegradable.
I
8t
Acknowledgments
We thank Prabhakar V. Arur, Tapan Chakrabarti, and Visvnath G. Joshi for useful discussions and comments. Registry No. AN, 62-53-3; NB, 98-95-3; water, 7732-18-5.
I
I
8
I
HOURS
I
16
I
2.4
Figure 5. Changes in pH following biodegradation of nitrobenzene (X), aniline (O), and a mixture of nitrobenzene and aniline (0).
the release of the ammonium ion. This has been qualitatively confirmed in the laboratory. Since aniline is toxic to nitrifiers in batch reactors (20),the released ammonium ion could not be nitrified as long as aniline remained in the system. However, nitrification started after 4 h of aeration, as is evident from the progression of the decrease in pH with time beyond 4 h. When the experiment was repeated with binary substrate containing aniline and nitrobenzene, the maximum pH observed was nearly equal to that of aniline in monosubstrate, whereas the rate of pH change was faster in binary substrate. This may be possibly due to the fast release of ammonium ion from aniline in the presence of nitrobenzene, which is acidic in nature. However, the removal rates are nearly equal.
Conclusions The present investigation leads to the following conclusions. (1)The direct injection GC method has proved to be practicable, accurate, and rapid. (2) Biodegradation rates are nearly equal for monosubstrate, binary substrate, and waste water system. (3) The substrate(s) removal is complete and simultaneous, and the components do not interfere mutually. (4) The relative proportions of two components in the range studied do not affect the rates of biodegradation.
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