Environ. Scl. Technol. 1988.22, 1160-1165

seasonal changes on the accumulation of HzOZ in a natural water.

In the environment, the profiles of Hz02 accumulation 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 H20z down 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 H202 is related to the concentration of these natural substances and appears to be ubiquitous in its occurrence in natural waters. From the experimen- tally 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,

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.

220,711-2.

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

Zepp, R. G.; Skurlatov, Y. L.; Pierce, J. T. In Photochem- istry 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.

250-67.

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 devel- oped 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

industries manufacturing synthetic resins, pesticides, dyestuffs, drugs, photographic chemicals, varnishes, vul- canization accelerators, and antioxidants. They are dis- charged 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 ir- ritation; affects the central nervous system; and produces fatigue, headache, slight dizziness, vertigo, and vomiting (2). Its continuous exposure leads to liver damage,

1160 Environ. Sci. Technol., Vol. 22, No. 10, 1988 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, re- spectively. The 6-h LCso for 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 en- vironmental 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 puri- fication 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 di- nitrophenol 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 m- aminophenol was complete whereas rn-nitrobenzene- sulfonate could be removed only partially. Austern et al. (14) have used extraction with freon and a gas chromato- graphic 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 mu- nicipal and industrial waste water discharges. GC has also been used for detection and determination of other aro- matic compounds like phenols, cresols, xylene, toluene, and benzene in waste waters (16, 17).

In this paper we report a systematic study on the bio- degradation of aniline and nitrobenzene in monosubstrate, binary substrate, and aniline plant waste water system by flame ionization gas chromatography using a Tenex col- umn. 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 inves-

tigation was obtained from a sewage treatment plant treating domestic sewage on the extended aeration prin- ciple. 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, dodeca- hydrate (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 fill- and-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 mon- itored 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 ac- climated when it reduced the waste water COD content to the extent of around 90-9590. The period of acclima- tization was 15 days.

Monosubstrate and Binary Substrate Biodegrada- tion. Bench-scale batch bioreactors I and I1 using accli- mated activated sludge were employed to study the biod- egradability of aniline, nitrobenzene, and a mixture of aniline and nitrobenzene. The biodegradation systems include monosubstrate containing either aniline or nitro- benzene 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 men- tioned earlier. For each system a control containing sub- strate(s) and nutrients was maintained to determine the extent of substrate removal by air stripping. The bior- eactor 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, nitro- benzene, 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

parameter min max av SD

total solids 140 190 163 17 COD 4554 8350 6007 1355 nitrobenzene 1520 2010 1828 159 aniline 650 2105 1230 502

PH 8.1 10.0

"Results are the average of eight sets of composite waste (each set collected on different days). All values except pH are ex- pressed as mg

Table 11. Recovery of Aniline (AN) and Nitrobenzene (NB) Spiked into Tap Water (Amounts in mg L-I)

I:

0 10 TIME (min 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 in- jected directly into the gas chromatograph. Relative re- sponse factors were determined for both aniline and ni- trobenzene 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 bio- degradation rate run.

Results and Discussion Characteristics of waste water from the aniline manu-

facturing plant are shown in Table I. A typical chroma- togram obtained from 2 pL of an aqueous solution con- taining 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

set

1 AN NB

2 AN NB

3 AN NB

4 AN NB

5 AN NB

6 AN NB

7 AN NB

8 AN NB

9 AN NB

amount taken

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

amount found

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

% error

-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

0 10 TIME (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 pos- sible losses.

The disappearance of peaks of aniline and nitrobenzene from the monosubstrate, binary substrate, and waste water

I 10 TIME i m i n )

L I 10

TIME (mi")

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)

amount amount coeff of set taken found' SD variance, %

I AN 27.9 27.8 0.1 0.4 NB 30.6 31.5 0.1 0.4

I1 AN 106.8 107.2 1.5 1.4 NB 118.5 118.2 3.4 2.9

I11 AN 320.4 322.0 2.2 0.7 NB 355.4 355.9 4.0 1.1

'Average of five determinations.

B

10 i I M E ( m i n ) l i M E ( m i n ) : ' ~ E f n t n

Flgure 4. Chromatograms showing biodegradation of aniline piant waste water system containing (B) aniline (C) nitrobenzene, and (A) CHA.

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, re- spectively, whereas traces of aniline (Figure 3) and nitro- benzene (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 bio- degradable in presence of each other. The metabolic in- termediate in the biodegradation process was not traceable

Table IV. Biodegradation of Aniline (AN)

control bioreactor I," 1 = 0.05 g of AN

(E! of MLVSSP bioreactor 11," 1 = 0.048 g of AN

(E of MLVSS)-' I .-

AN COD AN COD AN COD remaining, remaining, remaining, remaining, remaining, remaining,

aeration time, h mg L-' mg L-' mg L-l mg L-' mg L-' mg L-' 0 215 493 2 212 490 4 216 492 6 216 494 7 214 492

MLSS, mg L-' MLVSS, mg L-' ultimate biodegradation rate,

mg COD removal g-' MLVSS h-l

215 493 175 398 108 245

5 61 nil 47

5810 4270

17

215 493 178 405 109 247

7 68 nil 54

6080 4500

16

a 1 = loading. Results are the average of three experiments for each bioreactor.

Table V. Biodegradation of Nitrobenzene (NB)

bioreactor I," 1 = 0.043 g of NB

NB COD NB COD NB COD remaining, remaining, remaining, remaining, remaining, remaining,

bioreactor 11," 1 = 0.041 g of NB control (g of MLVSS)-l (g of MLVSS)-'

aeration time, h mg L-' mg L-' mg L-' mg L-' mg L-l mg L-' 0 183 329 183 329 183 329 2 183 327 114 203 121 215 4 183 326 46 83 41 72 5 179 320 7 29 6 37

MLSS, mg L-' 5790 6080 MLVSS, mg L-l 4260 4500 ultimate biodegradation rate, 14 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)

bioreactor 1,' 1 = 0.052 g of AN + 0.052 g of NB (g of MLVSS)-' control

AN NB COD AN NB COD remaining, remaining, remaining, remaining, remaining, remaining,

aeration time, h mg L-' mg I,-' mg L-' mg L-' mg L-' mg L-' 0 223 222 911 223 222 911 2 223 221 914 179 162 705 4 225 224 913 118 101 455 6 224 223 910 7 32 73 7 222 223 912 nil nil 65 8 223 221 913 nil nil 55

MLSS, mg L" MLVSS, mg L-I ultimate biodegradation rate,

mg COD removal g-' MLVSS h-l

1 = loading. Results are the average of three experiments for each bioreactor.

5800 4260

33

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-'

223 222 911 187 155 708 134 85 460 63 29 198

7 nil 95 nil nil 59

6100 4530

26

Table VII. Biodegradation of Aniline Plant Waste Water Containing Aniline (AN) and Nitrobenzene (NB)

bioreactor I," 1 = 0.033 g of AN + 0.058 g of NB (g of MLVSS)-'

AN NB COD AN NB COD AN NB COD remaining, remaining, remaining, remaining, remaining, remaining, remaining, remaining, remaining,

bioreactor 11,' 1 = 0.034 g of AN + 0.060 g of NB (g of MLVSS)-' control

aeration time, h mg L-' mg L-' mg L-* mg L-l mg L-' mg L-' mg L-' mg L-' mg L-' 0 150 250 784 150 250 784 150 250 784 2 153 253 786 88 158 488 107 172 556 4 151 249 784 1 80 147 21 99 227 5 151 251 782 nil 40 73 nil 64 114 6 151 250 784 nil nil 39 nil 24 94

MLSS, mg L-I MLVSS, mg L-' ultimate biodegradation rate,

mg COD removal g-' MLVSS h-'

6080 4270

33

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)

bioreactor I bioreactor I1 bioreactor 11' degradation, degradation, rate of rate of

mg of Sb mg of S 1 = g of S degradation, 1 = g of S degradation (g of MLSS)-' (g of MLSS)-' (g of mg of S (g of (g of mg of S (go of

biodegradation system h-l h-' MLVSS)-l MLVSS)-l h-l MLVSS)-l MLVSS)-l h-l

AN monosubstrate 6.0 5.1 0.050 8.2 0.048 7.7 AN binary substrate 6.2 5.1 0.052 8.5 0.049 6.8 AN waste water system 6.1 5.7 0.033 8.7 0.034 7.7 NB monosubstrate 6.1 5.8 0.043 8.3 0.041 7.8 NB binary substrate 5.5 5.3 0.052 7.5 0.049 7.1 NB waste water system 6.9 6.7 0.058 9.8 0.060 9.1

bioreactor IC rate of rate of

Represents the primary biodegradation rates. bSubstrate. 1 = loading. Temperature = 26-28 O C .

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, re- spectively, 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 ul- timate biodegradation in terms of COD has also been given

1164 Environ. Sci. Technoi., Vol. 22, No. 10, 1988

in Tables IV-VII. Although the primary biodegradation of the substrates shows 100% removal, the ultimate bio- degradation 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 mo- nosubstrate 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 sys- tems 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 sub- strates. In the case of aniline, the increase may be due to

I I

8 t

I I I I I 8 16 2.4

HOURS 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 quali- tatively 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 con-

clusions. (1) The direct injection GC method has proved to be

practicable, accurate, and rapid. (2) Biodegradation rates are nearly equal for monosub-

strate, binary substrate, and waste water system. (3) The substrate(s) removal is complete and simulta-

neous, 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.

( 5 ) Change in pH due to 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 biode- gradable.

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.

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Received for review May 18,1987. Accepted March 23,1988. This work was supported by the Council of Scientific and Industrial Research, New Delhi.

Environ. Sci. Technol., Voi. 22, No. 10, 1988 1165