The objective of this investigation was to

determine the ability of specifically adapted bacteria

to degrade phenol, when used as sole source of

carbon in mineral salts medium at higher

concentrations than that are generally present in the

natural environment

Chandrika, V

Imelda, Joseph

CMFRI Digital Repository

Indian Journal o f  Marine Sciences Vol. 28, Decem ber 1999, pp. 438-442Biodegradation of phenol using bacteria from different brackishwater habitatsIm elda Joseph * &  V  Chandrika  Central Marine Fisheries Research Institute, Post Box 1603, Tatapuram P O, Cochin 682 014, Kerala, IndiaReceived 6  July 1998, revised  Aug 16 1999present bacteria could be o f great help in their biodegradation and purification o f the systemBiodegradation is a difficult process to be quantified since living organisms in a dynamic environment carries it out. The occurrence and degradation of phenolic compounds by bacteria, the mechanisms involved in the process, the catabolic pathways and intermediate products of degradation and optimal conditions required for the processes are studied by many workers1 4. Microorganisms that can oxidize phenols were also reported4'5. Due to high volatility, phenols impart bad taste and odour to the aquatic environment even at part/billion levels in some cases.A wide range of phenol concentration (0.08 to 1800 ppm) pose serious pollution problem, adversely affecting the organisms of food chain and fish population by interfering with carbohydrate, protein and lipid metabolism, ions transport, nerve conduction and energy production and at molecular levels due to oxidative phosphorylation in fishes ' .Desired level of phenol for the protection of public health is 3.5 ppm and the level of controlling undesirable taste and odour of ambient water is 0.3 ppm.Microorganisms posses a remarkable adaptive capacity and can develop resistance to many toxic compounds. But all of them are not able to degrade toxic compounds like phenol. Only some enzymes called constitutive enzymes are present in an organism in physiologically significant amount under all conditions. Others are formed as and when needed only 9. It is therefore necessary to adapt bacteria to♦Present address: NRC of CIBA, Narakkal, Cochin-682 505, Kerala, Indiathe compound introduced in the environment to facilitate the induction of the enzymes. Predominance of such bacteria in a mixed culture would effectively remove the pollutant from the environment .The objective of this investigation was to determine the ability of specifically adapted bacteria to degrade phenol, when used as sole source of carbon in mineral salts medium at higher concentrations than that are generally present in the natural environment.Materials and MethodsBacteria were isolated from a coconut husk retting ground, a mangrove swamp, a free flowing backwater system and seasonal and perennial aquaculture ponds. During the process of coconut husk retting, polyphenols are reported to be leached out into the ambient water, thus causing heavy organic pollution11. Many workers also report the presence of phenolic compounds in mangrove swamps ' ' .  Apart from natural phenolic compounds leached from plant degradation, synthetic compounds also occur in aquatic ecosystems from effluents of oil refineiies and other factories where phenolic resins aremanufactured l4.Method used in selecting or adapting bacteria to degrade phenol was by primary enrichment on mechanical shaker2 and further screening and quantitative study by static flask culture method '. One part of culture enriched by mechanical shaking were inoculated in 250 ml conical flasks containing 50 ml of fresh sterilized mineral salts mediumJOSEPH & CHANDRIKA: BIODEGRADATION OF PHENOL 4 3 9 ^ 5(MSM) containing 0.25 jig of vitamin B12 (Table 1).Phenol (100 to 800 ppm), o-cresol (100 to 500 ppm) ^ or orcinol (100 to 500 ppm) served as the sole source of carbon in the .medium. The pH  of the medium was } adjusted at 7.0 to 7.2. The culture flasks wereincubated at room temperature (28±2°C). At weekly  ^ intervals, sub culturing was done (Sub culture I, Sub. culture II and Sub culture HI). Triplicates of eachconcentration were maintained.) Bacterial cells were separated by centrifugation and the supernatant was filtered and used to estimate ) residual phenol calorimetrically following the4-amino antipyrene m ethod16. The mixed cultures 5 were pour plated on mineral salts agar (MSM + 1.5%j agar) and seawater agar. Seawater agar was used formaintaining the culture on slants. The colonies were > isolated and characterized using standardmorphological, physiological and biochemical tests ' . The presence of Pseudomonas spp was confirmed with the help of cytochrome oxidase test. The scheme used for identification was that of Simidu & A iso19.Two way ANOVA was done for analyzing statistical significance of biodegradability of phenol over three sub cultures and over concentrations20.ResultsEnrichment experiments and biodegradability studies have demonstrated that phenol could be significantly biodegraded. The rate of biodegradation ranged from 46 to 100% for the bacterial samples collected from four different locations (Table 2). When phenol was added up to 300 ppm, complete degradation was observed within three days of incubation. As expected, the rates of degradation showed significant increase with the sub culture and were highest for sub culture HI (Table 3).Phenol utilizing bacteria were present at all the four; stations sampled. They were mostly Gram negative, aerobic, and small to medium sized rods. Both motile and non-motile forms were present. Based on morphological and • biochemical characteristics, the colonies were tentatively identified as Alcaligenes, Cytophaga, Enterobacteriaceae, Flavobacterium, Pseudomonas, and Vibrio. Gram positive Bacillus sp. and Micrococcus sp. were also present in the samples.The percentage distribution of bacteria at different concentrations of phenol (100 to 800 ppm), o-cresol (100 to 500 ppm) are given in Table 4. At higherTable 1— Composition o f  mineral salts medium (M SM )k 2h p o 4 ■ — I.OgM g S 0 4' — 0.2  gNaCl — 15.0 gCaCI2 — O .IgFeCl2 — 0.02 g(NH4)2 s o 4 — I.OgDistilled water — 1.0 literPhenol — A>pH — 7 .0  to 7.2Table 2— Biodegradability o f  phenol using mixed culture o f  bacteria.[Average o f  triplicate (9 days)]Phenol Biodegradation (%)conc (ppm) IS C II SC III SC100 a) 100 100 100b) 100 100 100c) 100 100 100d) 100 100 100200 a) 100 100 100b) 100 100 100c) 100 100' 100d) 100 100 100300 a) 100 100 100b) 100 100 100c) 100 100 100d) 85 95 100400 a) 90 100 100b) 80 92 100c) 75 90 100d) 72 83 98500 a) 80 95 100b) 80 92 100c) 70 80 90d) 68 76 80600 a) 78 80 85b) 75 77 79c) 68 74 78d )* 5 70 73700 a) 74 78 80b) 70 73 78c) 65 69 76d) 63 66 68800 a) 60 66 48b) 48 54 60c) 48 52 54d) 46 50 52a) coconut husk retting area; b) mangrove swamp; c) backwater and d) aquaculture pondsconcentrations of phenol <500 ppm and above), only Pseudomonas and Vibrio were present. Most of the genera, which utilized phenol, were also present in o- cresol and orcinol. Two way ANOVA indicates that F4 4 0 INDIAN J. MAR. SCI., VOL., 28, DECEMBER 1999T able 3 — Two way ANOVA for biodegradability o f  phenol over sub cultures and over concentrationsSource Degrees o f Sum o fMean sum o f F Remarksj Freedom squares squares(A) Coconut husk retting areaSub culture 2 214.32 107.164.72 sig (5 %)Concentration 9 6410.30 712.26 31.57sig (5 %)Error 18 408.68 22.70Total 29 7033.30(B ) M angrpve swampSub culture 2 232.20 116.11 6.58sig ( I %)Concentration 9 7742.70 860.30 48.75s ig ( l  %)Error 18 37.6 17.65Total 29 8292.50(C) Back-water system  'Sifb culture 2 321.58 160.79 7.68sig (1 %)Concentration 9 8006.31 889.59 42.50sig (1 %)Error 18 376.81 20.93Total 29 8704.69(D ) Aquaculture ponds J.Sub culture 2 301.24 150.62 7.99sig (1 %)Concentration 9 7546.60 833.51 44.46sig  (1 %)Error 18 339.50 18.87Total 29 8187.31values for all the four stations between sub cultures and concentrations were significant at P<0.01 and P<0.05 respectively (Table 3).DiscussionEnrichment culture was done by shake flask culture technique for faster multiplication of phenol using bacteria and for better adaptation to phenol. For further studies, the static-culture flask biodegradation method was followed in the present investigation using mixed bacterial isolates from different locations. Bunch & Chambers21 and Tabak et al. followed a similar approach.The source of inoculum showed considerable influence on the biodegradation of phenol in the present study. Bacteria from the coconut husk retting area, with high load of organic wastes, showed maximum rate of biodegradation. Adaptation of bacteria to phenol had significant correlation with biodegradation. It was reported earlier that bacteria should be well adapted to metabolize phenol, hydroxyphenols etc., when cultured in MSM2. In the present study also, biodegradation studies were conducted after adapting the bacteria from different locations by primary enrichment technique. Also ondetermining biodegradation rates following static culture flask, it was found that many bacteria could tolerate phenol concentration as high as 800 ppm after proper adaptation. A similar observation was reported earlier in which the most adapted group as sub culture III in the present study utilized highest quantity of phenol15.It has been shown that 1000 ppm to 2400 ppm of phenol can be degraded by bacteria or fungi after proper adaptation1,3,11,12. In the present study, mixed bacterial populations degraded 100 ppm to 800 ppm of phenol. Phenol pollution is.not a serious problem at any of the locations sampled, however, the study is important if the aquatic environments get polluted by. Of different types of pollutant treatments are followed, biodegradation is preferred since it is economical and harmless4.The results indicate that phenol adapted bacteria could utilize up to 300 ppm of phenol in the medium within 9 days. It is also observed that the rate of biodegradation is dependent on the concentration of phenol in the medium (Table 2). It was interesting to note that maximum degradation of phenol in the initial two to three days and only marginal increase in the proceeding days. It was reported that rate ofJOSEPH & CHANDRIKA: BIODEGRADATION OF PHENOL 441Table 4 -P e r c e n ta g e  distribution o f bacteria at different concentrations o f  phenol, o-Cresol and orcinol in biodegradation experimentsPhenol conc (ppm) ____100 200 300 400 500 600 700800A lca ligen es 18.8 12.5 14.3 12.5 16.6 — —---B acillus 9.1 — — -- — -C ytophaga 8.7 — — -- ---E nterobacteriac 9.1 12.5 14.8 12.5 16.7eaeF lavobacterium 8.9 12.5 — — — --- —M icrococcus 9.1 12.5 — 28.6 12.5 16.7 — ---P seudom onas 27.3 25 28 37.5 33.3 33.4 5066.3Vibrio 9 25 14.3 ' 25 16.7 66.6 5033.7o-Cresol conc (ppm)100 200 300 400 500A lcaligen es 15.3 — — — —B acillus 7.6 — — — —C ytoph aga 7.9 — — — —Pseudom onas 46.1 77.4 66.7 80 82Vibrio 23.1 22.6 33.3 20 18Orcinol conc (ppm)100 200 300 400 500A lcaligen es 10.0 — — — —B acillus 30.0 14.5 — — — >C ytoph aga 10.0 6.6 4.3 — —F lavobacterium 10.0 13.0 12.3 6.7 6.7P seudom onas 20 .0 42.6 40.6 40.0 37.3Vibrio 20.0 23.3 42.8 53.3 56.0biodegradation to be more during the first phase of incubation period2.Phenololytic microorganisms belong to bacteria, fungi and actinomycetes. Among bacteria, those belonging to genera, Azotobacter, Alcaligenes, Pseudomonas, Flavobacterium, Clostridium, Micrococcus, Vibrio, Sarcina^etc. were reported to utilize phenolics as sole source of carbon 1 . In the present study, bacterial genera, which could tolerate phenol at different concentrations, were Alcaligenes, Bacillus, Cytophaga, Enterobacteriaceae,Flavobacterium, Micrococcus, Pseudomonas and Vibrio. Same genera were encountered in orcinol and oTcresol also. At concentrations above 400 ppm only Pseudomonas sp. and Vibrio sp. were present.The chemical structure and concentration of the substrates are important considerations in biodegradation process23. Though chemical and some biological processes bring about some changes in the chemical structure of the compound introduced (phenol) thereby reducing the risk of environmentalpollution. Thus it forms an effective method of pollutant treatment in environment when other processes are not economically feasible and socially acceptable.AcknowledgementThe first author is thankful to the Indian Council of Agricultural Research (ICAR), New Delhi, for the financial assistance provided during the period of this study.References1 Gray P H H & Thornton H G, Z m te r  B acteria l Parasitenk, Abstract II (1928) 73.2  Tabak H H, Chambers C W & Kabler P W, J B acteriol, 87 (1 9 6 4 )9 1 0 .3 Itturiaga R & Rheinheimer G, K eiler M eeresforschung, 28 (1 9 7 2 )1 .4  Shivaraman N & Parhad N M, Indian J  M icrobiol, 25 (1985) 79.5 Aaronson S, in Experimental m icrobial eco logy  (Academic Press, New York & London). 1970, 65.6 Desiah, D , Pentachlorophenol, 1 2 (1 9 7 8 )2 7 7 .4 4 2 INDIAN J. MAR. SCI., VOL., 28, DECEMBER 19997 Gupta, S , in P roceedings o f  sym posium  on assessm ent o f  environm ental po llu tion  (The Academ y o f  Environmental B iology, M uzaffamagar India) 1985, 151.8 Anon, A m bient w a ter quality  criteria  f o r  phenol, EPA  440/5- 0-066  (United States Environmental Protection Agency), 1980. ’9 Slater J H & Som erville H J, in M icrobial technology, edited by A  T  Bull, D  C Ellw ood & R Ratledge (Cambridge Uni Pi£ss, Cambridge) 1979, 128.10 Sastry C  A , Encology, 1 (1986) 29.11 j ° l e p h ’ l ' in  BiodeS rada tion  o f  pheno lic  com pounds in d ifferen t ecosystem s in  C ochin, Ph. D, thesis, Cochin University o f  Science and Technology, India, 1997.’ ^  4  nQ 7S N  G’ L° kbharathy P A  & Nair s > M ia n  J  M ar Sci,■ \ I 975) 215« 113 G om es H P  &  Mavinkurve S, M ahasagar—Bull N atl Inst Oceanogr, 15 (1982) 111.14 Jorgenson S E, Vatten, 27 (1971) 434.15 Tabak H H, Quave A  S, Mashmi I C &  Barth F E, J  Water P ollut C ontrol Fed, 53 (1981) 1503.16 APHA, in Standard m ethods f o r  examination o f  w ater an d  w aste water, (ed 14) (American Public Health Association Inc, N ew  York) 1975, 574.17 Gaby W L & Hadley C ,J  B acteriol, 74 (1957) 356.18 Gaby W  L  & Free E, J  Bacteriol, 7 6 (1 9 5 8 )4 4 2 .19 ^  & AiS° ^  BUl1 ^  S°C SC' FiS,t" 28 ^1962)20 Snedecor G W &  Cochran W  G, in S tatistical methods, (Oxford & IBH Publ Co, N ew  Delhi) 1967, 593.21 39 0 9 6 7 ) GhamberS C  W ’ J  W a te r  P o l l l l t  C o ,U r o t22 Visser S A, Lamontagne G, Zoulalian V & Tassier A, Environ Contain Toxicol, 6 (1977) 455.23 Bourquin A W, in G enetic control o f  environm ental pollu tan ts, edited by O S Gulbert & H Alexander, (Plenum  Press, N ew  York), 1984,97.i-cientistfc0ebia.682MH( Indij,

Biodegradation of phenol using bacteria from different brackishwater habitats

438-442Bacterial
degradation of phenol (100 to 800 ppm) was studied. It was observed that
bacterial diversity and the rate of phenol biodegradation were influenced by
the phenol concentrations in the medium. The rate of biodegradation was reduced
at higher concentrations of phenoli and Pseudomonas spp and Vibrio
spp were mainly involved in the process. The distribution of bacterial
genera in the medium was dependent on the compound and its concentration. Pseudomonas
and Vibrio were present in all concentrations of phenol, o-cresol
and orcinol. Whereas, Bacillus and Alcaligenes could tolerate
only up to 200 ppm of these compounds in the medium.  Flavobacterium was absent in o-cresol,
while it could tolerate 500 ppm of orcinol and 200 ppm of phenol. Cytophaga tolerated
300 ppm of orcinol, and 100 ppm of phenol and o-cresol.

From the
present study, it is concluded that in case of an accidental spill of these
phenolic compounds many naturally present bacteria could be of great help in
their biodegradation and purification of the system thereafter

Biodegradation of phenol using bacteria from different brackishwater habitats

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