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 Col lege, 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 accl im�lted activated sludge in a laboratory scale batch bioreactor. By gaschromatographie (GC) monitoring i t is found that the uti l ization of anone and anol are sequential and not s imultaneous. Anol is uti l ized only after anone gets exhausted by about 90 per cent. When the experiment is repeated with anol as a mono substrate i t is uti l ized instantaneously without any l ag period. The effect of sustrate loading and Mixed Liquor Suspended Solids (MLSS) on sequential degradation has also been reported. I t is concluded that anone is preferen­t ia l ly 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 iodegradabi l i ty of din i trophenols t , n itrobenzene2, aminophenoP, ni trobenzene, and an i l ine4 was studied earl ier using acc l imated activated sludge. B i odegradabi l i ty 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 l ity.

Ti l l now no work has been reported on the bio­degradation 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 detai l , 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 artific ial wastewater was

s imulated 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 s ludge was col­lected from a run ning wastewater treatment p lant of a caprolactam manufacturing unit. Within 0.5 h the s ludge was brought to laboratory and al l owed to settle for 0 . 5 hours and the supernatant was with­drawn. The settled s ludge was washed with tap wa­ter several t imes and used for the accl i mation pro­cess.

Reactor-The reactor was fabricated in the l abora­tory. It is made of g lass, cyl indrical 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 esti­mation of anone and ano l w as c arried out in a Hewlett Packard Gaschromatograph model 5890.

J

BABU & VERllHESE : CYCLOHEXANONE & CYCLOHEXANOL 865

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 contain ing 200 mg/I of each in dis­til led water. It was calibrated in GC by external stan­dard method. The anone and anol were suppl ied by MERCK, Germany.

Acclimation of Activated Sludge

The sludge was accl imated with artificial waste­water contain ing 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 in­volved 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 requi red for any activated sludge process8. The pH was main­tained 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 with­drawing calculated amount of sludge from the reac­tor and replacing with the equal amount of water. The accl imation process included 8 h aeration, set­tling 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, di ssolved oxygen, sludge volume, conductivi ty, Ammonia-Nitrogen, P, COD and MLSS were carriedout. The accl ima­tion process was completed i n 5 d.

Experimental Procedure

Accl imated 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 accl imation process. Samples were withdrawn at regular interval s from the bioreactor and used for GC and chemical analysis . The sludge was periodically monitored for pH, DO, sludge vol­ume, ammonia nitrogen (N), COD, phosphorous (P) and MLSS (Mixed Liquor Suspended Sol id). In all the three experiments the aeration was cut after 8 h, al lowed to settle for 0.5 h, supernatant was with­drawn, and settled sludge was washed with tap wa­ter twice and made up to the working volume ( 1 01) and used for the next cyc le of operations. For each experiment, appropriate controls were carried out without activated s ludge to check the extent of sub­strate loss due to ai r strippint. A graph was plotted in the al l 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 GI 0::

o Incu bat ion 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 ac­climated seed in the B OD bottles at 20°e. Five sets of samples with same di lutions were kept for incu­bation. After every 24 h the residual DO was deter­mined by Winkler method. The experiment was continued for 5 d. Same number of seed controls were also run simultaneously to check the seed con­sumption of DO. Graphs were plotted in each case (Figure 1 ) with residual DO in mg/1 against incuba­tion 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 uti l ized by the microorganisms within 5 h of aeration under the experimental con­di tions. 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

..... ..... 2 00 01 E . ., ... 1 5 0 c: � 0 E 0 01 C 'c 'Cj E <11. 0::. 0 2 3 t. 5

Aerat ion 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 QI a::

0 2 3 4 5 A �rat i on t i me , h

Figure 2(b)-RDO level during • degrad ation o f anol as monosubstrate. � sequential degradation of anone and anol

GC analysis shows that anol was not util ized sig­nificantly up to 2 h and i t got consumed by the mi­croorganisms only after the anone was exhausted by about 90 per cent . Further, anol degraded rap­idly. But i n the experiment with anol alone, as a mono substrate, it was found that anol was consumed instantaneously. Thi s observation confirms with earl ier report on the sequential degradation of glu­cose and lactate 10, nitrobenzene and ani l i ne4. The degradation of anone was not affected by the pres­ence of anol in any of the experiments The corre­sponding graph showing degradat ion pattern c anone and anal as binary substrate and mono Sl strate i s shown in Figure 2a. Figure 2b represe

BABU & VERGHESE : CYCLOHEXANONE & CYCLOHEXANOL 867

the residual DO level corresponding to different durations in the above experiments.

In order to explain the reason for sequential uti l i­zation of anone and anol the fol lowing theories were proposed. When certain parameters become l imit­ing, competition among microorganisms may take place and that may lead to sequential util ization of the substrates. These factors may either be nutrient concentrat ion, dissolved oxygen content of the sludge or may be due to difference in oxygen up­take rate. In order to check whether nutrient is l im­iting, N and P were suppl ied in large excess than that was required and the experiment was repeated and found that there was no change in the degrada­tion pattern .

The Oxygen Uptake Rate (OUR) by the microor­ganisms 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 differ­ence 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 acti­vated 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 d isproves 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 me­tabol ism of anol . But it is already established that the first step in the metabolic pathway of anol deg­radation is the formation of anone l2. In our experi­ments with anol as a mono substrate the anone that was formed by the metabol ism of anol did not in­hibit degradation of anol . Hence, inhibitory effect

of an one was not considered as a reason for the se­quential degradation of the substrates. As described earl ier the first step in the pathway of anol degrada­tion is the formation of anone, i .e, anol was oxi­dized to anone before being metabolized in to lower organic acids. Thus, as long as anone is avai lable in the system the microbes will preferentially attack the more easily degradable substrate, which in the present case is anone. Anol wi l l be utili zed only when there is not enough anone is in the system.

In the experiment with different substrate load­ing and MLSS it was found that by increasing the Food/Microorganism mass (F/M) ratio, the sequen­tial degradation pattern was not affected. But the time required for complete removal of the sub­strate was found to increase with h igher F/M ratio (Table 1 ) .

The biodegradabil ity 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 b iodegradable by using an accl imated activated sludge from a caprolactam wastewater treatment unit . Uti l ization of cyclohexanone and cyclohexanol by the microorganisms is not simul­taneous. 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 re­quired 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 Pi l lai 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 Tech­nology for useful discussions and comments .

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