Journal of Scientific & Industrial Research Vo1.58. November 1 999, pp 864-868
Biodegradation of Cyclohexanone and Cyclohexanol by the Activated Sludge Process
G Babu and Winny Varghese*
Department of Chemistry, Mar Athanasius College, Kothamangalam, Kerala 686 666, India Received: 22 June 1 999; accepted: 23 August 1 999
Biodegradability study of cyclohexanone (anone) and cyclohexanol (anol), two major organic pollutants present in the wastewater from any caprol" ctam manufacturing unit which fol low a Raschig process, is carried out with an artificial eftluent using an acclim�lted activated sludge in a laboratory scale batch bioreactor. By gaschromatographie (GC) monitoring it is found that the util ization of anone and anol are sequential and not simultaneous. Anol is utilized only after anone gets exhausted by about 90 per cent. When the experiment is repeated with anol as a mono substrate it is utilized instantaneously without any l ag period. The effect of sustrate loading and Mixed Liquor Suspended Solids (MLSS) on sequential degradation has also been reported. It is concluded that anone is preferen
tially consumed from a binary mixture of anone and anol by the mi xed population of microorganisms i n an activated sludge under the experimental conditions.
Intoduction
Several studies have already been done with ac
tivated sludge process in the treatment of domestic as wel l as industrial wastewater. B iodegradabil ity of dinitrophenols t , n itrobenzene2, aminophenoP, nitrobenzene, and an iline4 was studied earl ier using acclimated activated sludge. Biodegradability of caprolactam wastewater contai ning cyclohexanone and cyclohexanol and cyclohexanone oxime has been studi ed) and it is reported that 90-95 per cent BOD reduction has been ach : . .::ved. Cycl ohexanone and cyclohexyl am ine con t aining wastewater6 was studied on their biodegradabi lity.
Ti l l now no work has been reported on the bio
degradation of anone and anol in the wastewater from a caprolactam manufacturing unit by the acti
vated sludge process. The objective of the present work was to study, in detail, biodegradation of anone and anol and to study the effect of FIM ratio (Food/
Microorgani sm) on the degradation pattern.
Materials and Methods
Wastewater Source-An artificial wastewater was simulated in the laboratory by dissolving calculated
* Author for correspondence
amount of cyclohexanone and cyclohexanol in tap water.
Activated Sludge-Ten l i ters of the sludge was col
lected from a run ning wastewater treatment plant of a caprolactam manufacturing unit. Within 0.5 h
the sludge was brought to laboratory and all owed to settle for 0.5 hours and the supernatant was with
drawn. The settled sludge was washed with tap wa
ter several times and used for the accl i mation pro
cess.
Reactor-The reactor was fabricated in the l abora
tory. It is made of glass, cylindrical in shape having a capacity of 1 2 I and a motor driven agitator was provided at the center of the reactor. Conti nuous aeration was given through stone diffusers at a flow rate of 90 IIh .
CheTnical Analysis-Al l the chemical analysis were carried out by standard methods? (APHA 1 976).
Gaschromatography (GC)-The quantitative esti
mation of anone and ano l w as c arried out in a Hewlett Packard Gaschromatograph model 5890.
J
The following GC conditions were maintained.
Injector temperature : 200" C Detector Detector 200" C Carrier Gas Oven 1 60" C Flow (mllmin)
Column: Glass
: FlO : Nitrogen : 30
Stationary phase Carbo wax 1 0 per cent Supported gas chrom
Q.
Dimension Length 3m. 00 l,4the, 10; 4mm.
Calibration mixture was prepared with standard anone and anol containing 200 mg/I of each in dis
tilled water. It was calibrated in GC by external stan
dard method. The anone and anol were supplied by MERCK, Germany.
Acclimation of Activated Sludge
The sludge was acclimated with artificial waste
water containing anone and anol in the batch bioreactor. S ludge was taken in the reactor up to a working volume of 1 0 I. Acclimation process in
volved initial addition of artificial wastewater in such a quantity to get a concentration of 50 mgl l each of anone and anol in the reactor. The sludge was continuously aerated by admitting compressed air at a flow rate of 90 IIh to maintain a dissolved oxygen (DO) level of 3-4 mg/I which is required for any activated sludge process8. The pH was main
tained between 6-7 by periodic addition of sodium hydroxide solution. Phosphoric acid and ammonium sulphate were added as a source of nitrogen (N) and phosphorus (P), respectively, in such an amount to keep the nutrient ratio, COD:N:P at 1 00:5: I (ref.9).
The MLSS was maintained at 3000 mg/I by with
drawing calculated amount of sludge from the reac
tor and replacing with the equal amount of water.
The acclimation process included 8 h aeration, set
tling for 0.5 h, supernatant was withdrawn and the settled sludge was washed in tap water several times and made up to the working volume with water, and Ised for the next cycle. The experiment was repeated ith gradually increasing the loading of anone and 01 up to 300 mg/l each in the reactor. The biom-
ass was said to be accl imated when it could reduce the soluble Chemical Oxygen Demand (COD) to the extent of 90-95 per cent. During the accl imation process analysis such as pH, dissolved oxygen, sludge volume, conductivity, Ammonia-Nitrogen, P, COD and MLSS were carriedout. The accl ima
tion process was completed i n 5 d.
Experimental Procedure
Acclimated activated sludge was exposed to the artificial wastewater containing:
(a) Both anone and anol at a substrate loading of 0.07g anone and 0.07g anol/g MLSS
(b) Anone at a substrate loading of 0.07 g anone/
g MLSS
(c) Anol at a substrate loading of 0.07g anol/g MLSS
The pH, DO and nutrient ratio were maintained as given in the acclimation process. Samples were withdrawn at regular intervals from the bioreactor and used for GC and chemical analysis. The sludge was periodically monitored for pH, DO, sludge vol
ume, ammonia nitrogen (N), COD, phosphorous (P) and MLSS (Mixed Liquor Suspended Solid). In all the three experiments the aeration was cut after 8 h, allowed to settle for 0.5 h, supernatant was with
drawn, and settled sludge was washed with tap wa
ter twice and made up to the working volume ( 1 01) and used for the next cycle of operations. For each experiment, appropriate controls were carried out without activated sludge to check the extent of sub
strate loss due to air strippint. A graph was plotted in the all three experiments with the remaining amount of substrates and DO against aeration time.
The experiment was repeated at different substrate loading and MLSS to study the effect of FIM ratio on the biodegradation of anone and anol .
Determination of Oxygen Uptake Rate (OUR) for the Biodegradation of Anone and Ano)
The oxygen requirement for biodegradation of ( I ) Anone (2) Anol, and (3) Mixture of anone and anol
866 J SCI IND RES VOL.58 NOVEMBER 1 999
8
-L..
6
CI E
"0 0
'0 :l
:E en
0:: GI
o
Incu bation period , days
Figure I -Shows D 0 consumption for the biodegradation of 0 anone, 0 anol, t. mixture of anone and anol by standard BOD method
were determined separately by standard Biological Oxygen Demand (BOD) method. Suitable dilutions of each were predetermined and incubated with ac
climated seed in the B OD bottles at 20°e. Five sets of samples with same dilutions were kept for incu
bation. After every 24 h the residual DO was deter
mined by Winkler method. The experiment was continued for 5 d. Same number of seed controls were also run simultaneously to check the seed con
sumption of DO. Graphs were plotted in each case (Figure 1 ) with residual DO in mg/1 against incuba
tion time in days. In each experiment the oxygen uptake rate was calculated from the slope of graph.
The concentration of substrates corresponding to 0 h in the BOD bottle with mono substrate was, anone 3.3mg/l; anol 3 .7mg/1 and that for binary substrate was anone 1 .6mg/l ; anol 1 .8mgll .
Results and Discussions
Monitoring of GC analysis showed that anone and anol arc completely utilized by the microorganisms within 5 h of aeration under the experimental con
ditions. However the GC analysis of the controls, that was maintained without activated sludge and with continuous aeration for 5-8 h, showed negl i
gible removal of substrates by air stripping. The
...
... 01 2 00 E . .,
... c: 1 5 0
� 0
E 0 01 C
'c 'Cj E <11.
0::.
0 2 3 t. 5
Aeration t i me, h
Figure 2(a)-Sequential degradation of binary substrate containing t. anone and 0 anol, Substrate loading (I) O.07g anone, O.07g anol /g MLSS, MLSS = 2500 mg! l .
...
(]I
• Anol degradation as a monosubstrate. Substrate loading (I) O.058g anol! g MLSS. MLSS = 3000mg/ I
8
E 6
0 0 4 c; ::l
'0 2 'iii
a:: QI
0 2 3 4 5
A �ration ti me , h
Figure 2(b)-RDO level during • degradation of anol as monosubstrate. � sequential degradation of anone and anol
GC analysis shows that anol was not utilized sig
nificantly up to 2 h and it got consumed by the mi
croorganisms only after the anone was exhausted by about 90 per cent. Further, anol degraded rap
idly. But i n the experiment with anol alone, as a mono substrate, it was found that anol was consumed instantaneously. This observation confirms with earl ier report on the sequential degradation of glu
cose and lactate 10, nitrobenzene and aniline4. The degradation of anone was not affected by the pres
ence of anol in any of the experiments The corre
sponding graph showing degradation pattern c
anone and anal as binary substrate and mono Sl
strate is shown in Figure 2a. Figure 2b represe
the residual DO level corresponding to different durations in the above experiments.
In order to explain the reason for sequential utili
zation of anone and anol the following theories were proposed. When certain parameters become l imit
ing, competition among microorganisms may take place and that may lead to sequential utilization of the substrates. These factors may either be nutrient concentration, dissolved oxygen content of the sludge or may be due to difference in oxygen up
take rate. In order to check whether nutrient is l im
iting, N and P were supplied in large excess than that was required and the experiment was repeated and found that there was no change in the degrada
tion pattern.
The Oxygen Uptake Rate (OUR) by the microor
ganisms for the biodegradation of anone and anol was determined by B OD method. From the slope of the graph (Figure I ) OUR for the biodegradation of either anone, anol or mixture of both was obtained as 0. 1 5 mg/lih. This shows that there is no marked difference between the substrates in their OUR. Thus the sequential degradation that may due to differ
ence in OUR is ruled out in this case.
The residual DO was maintained between 2.0 and 3 .0 mg/I throughout the experiment, well above the critical DO content8 that was required for any acti
vated sludge process. From this, competition that may be due to l imiting DO content was also ruled out. Magorl l has shown that, anone was converted to anol in the cyclohexane degradation pathway by certain microorganisms. But in our experiments with anone anol as a mono substrate, no accumulation of anol in the system which disproves the formation of anol by the degradation of an one was found. The sequential degradation observed in our study may l ikely be due to the inhibitory effect of anone on the anol oxidative enzyme, which may arrest the me
tabolism of anol. But it is already established that the first step in the metabolic pathway of anol deg
radation is the formation of anonel2. In our experi
ments with anol as a mono substrate the anone that was formed by the metabolism of anol did not in
hibit degradation of anol . Hence, inhibitory effect
of an one was not considered as a reason for the se
quential degradation of the substrates. As described earlier the first step in the pathway of anol degrada
tion is the formation of anone, i .e, anol was oxi
dized to anone before being metabolized in to lower organic acids. Thus, as long as anone is available in the system the microbes will preferentially attack the more easily degradable substrate, which in the present case is anone. Anol will be utilized only when there is not enough anone is in the system.
In the experiment with different substrate load
ing and MLSS it was found that by increasing the Food/Microorganism mass (F/M) ratio, the sequen
tial degradation pattern was not affected. But the time required for complete removal of the sub
strate was found to increase with higher F/M ratio (Table 1 ).
The biodegradability observed by GC and COD methods show primary and ultimate biodegradation respectively. Although primary biodegradation of substrates shows 1 00 per cent removal the ultimate biodegradation in terms of COD is in the range of 90 per cent (Table I ).
Conclusion
Cylohexanone and cyclohexanol are found to be completely biodegradable by using an acclimated activated sludge from a caprolactam wastewater treatment unit. Utilization of cyclohexanone and cyclohexanol by the microorganisms is not simul
taneous. Cyclohexanol degradation starts only after cylohexanone has been exhausted by around 90 per cent. The sequential degradation pattern wr.s not affected by changing the F/M ratio, but the time re
quired for complete removal of substrates was found to increase with higher FIM ratio
Acknoledgement
The authors are grateful to Dr N K Pillai, Joint General Manager, FACT (Petro Chemical Division), Udyogamanda\, Kerala, Dr V N Sivasankara Pillai and Dr I S B rightsingh , School of Environmental
868 J SCI IND RES VOL.58 NOVEMBER 1 999
Table I -Effect of MLSS and F/M ratio on sequential degradation of anone and anol (substrate loading)
Aeration Remaining amount COD COD
time(h) anone anal remaining reduction
0 0.5 I -0.077 g an one, 0.077 g anol/g 1 .0
MLSS 1 .5
MLSS - 1 300 mg II 2.0
F/M-0.46 kg COD/kg MLSS 2.5
3.0 3.5
0.0 1 - 0.025 g anone, 0.025g anol/g 0,25
MLSS- 0.50
MLSS - 4000 mgl J 0.75
FIM - 0. 1 6 kg COD /kg MLSS 1 .0 1 .25 1 .50 0 1-0.0 1 7g anone, 0.01 7g anoJ/g 0.25
MLSS 0.50
MLSS - 3000 mg/ l 0.75
FIM - 0. 1 1 kg COD /kg MLSS 1 .00 0
1-0.033g anone, 0.066g anol/g 0.5
MLSS 1 .0
MLSS - 3000 mg/ I 1 .5
F/M - 0. 1 1 kg COD Ikg MLSS 2.0 2.5 2.,75
Studies, Cochi n University of Science and Tech
nology for useful discussions and comments.
References
Pitter P, Palyza, R & Hubnerova K H, Technol Toda);. F19( 1 974) 77-98.
2 Janezo, A & Gaz W, Tech San it, 58 (4) ( 1 984) 1 07- 1 1 1 . 3 Desh Pande S D, Chakrabarthy T, Subrahmanian P V R &
S underasan B B , Wat Res, 19 (3)(1 985) 293-298.
4 Pati! S S & Shindae V M. Eviron Pollut, 57 ( 1 989) 235-250.
5 Patel M D & Patel D R Indian J Environ Health, 19(4) ( 1 977) 3 1 0-3 1 8.
(mg/l) (mg/l) per cent
1 00 1 00 600 0
75 1 00
50 1 00
20 1 00
5.0 75
2.0 25
1 .0 5.0
0.0 0.0 70 88
1 00 1 00 620 0
75 1 00
45 1 00
1 8 1 00
2.0 75
1 .0 5 .0
0.0 0.0 80 87
50 50 320 0
1 5 50 220
2.0 1 8
1 .0 2.0
0 0 40 87.5
1 00 200 900 0
65 30 2.0 1 .0 0 0
200 1 99
1 75 600
1 1 0 60
0 90 90
6 B ieszkiewics E & M ajcher Z, Acta Microbial Polan, 42( 1 )( 1 993) 83-92.
7 APHA-AWWA-WPCF.Standard Methodfor the Examination )I' of Water and Wastewater, 1 4th ed, (American Public Health As
sociation, New York) 1 976.
8 Bennet G F & Kemp L L, Wat Reuse, Chem Eng Prog Symp 50;
63 (No.78)( l 967) 1 7 1 - 1 76.
9 B ates M H & Torabian A. War Res (G B), 15( 1 98 1 ) 999.
1 0 Lee I H , Fredrickson A G & Tsuchiya H M , Appl Micro Bioi, 28 (5) ( 1 974) 3 1 0-3 1 8-
I I M agor, Appl Environ Microbial, 52( 1 986) 665-67 1 . 1 2 Anderson Murray S,Hall Roberta A & Griffin Martin, J Gen
Microbial, 120( I ) ( 1 980) 89-94.
