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Issue 1 - 2000/01 |
ISSN 1311-8978 |
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EVALUATION OF THE BIOREMEDIATION ACTIVITY OF THE IDIGENOUS-MICROFLORA IN ADAPTATION PONDS OF THE PETROLEUM RAFINERY “LUKOIL” BOURGAS Asenova М. Milenova*, Jana I. Topalova*, Dimitar S. Kozuharov*, Raycho. I. Dimkov*, Chris Van Ceer** *Faculty of Biology, University of Sofia , “St.Kl.Ohridsky”, 8 “Dragan Tzankov” bld 1964 Sofia , Bulgaria, E-mail:topalova@biofac.uni-sofia.bg; masenova@biofac.uni-sofia.bg Fax: +359 2 65 66 41 ** CBRC, KaHo Sint –Lieven, Gebr. Desmetstraata 1, 9000 –Gent, Belgium, Fax: +32 9 265 86 38 E-mail: chris.vankeer@kahosl. Received: 25.07.01; Cited:18.10.01
Abstract The investigation is oriented to the adaptiv ponds of refinery – “Lukoil”, Bourgas. This zone (natural water treatment system) is buffering the influence of effluent- waters, from wastewater treatment plant, on the sea and performance an important ecological role. The accent is put on an investigation of the bioremediation activity of the indigenous micro-flora. The adequate and correct assessment of bioremediation activity will give an opportunity to control this activity. We suggest an investigation program for assessment of the flexibility in the bioremediation activity and the rate of biodetoxification in 10 sampling points, situated in the way of water flow, from 1-st to 4-th lakes. We started an investigation of the biodetoxification potential by means of following classical and advanced parameters and their combination – chemical and physical (COD, dissolved oxygen, temperature, pH and total suspended solids), microbiological (total bacterial count, quantity of aerobic heterotrophic bacteria and phenol-degrading bacteria), enzymological (dehydrogenase activity). The results showed that the applied approach in the bioindication is suitable for determination and management of the bioremediation activity of the microbial communities in water. Key words: Bioremediation, Hydromicrobiological Assessment, Dehydrogenase Activity Indigenous Micro-flora
INTRODUCTION The investigation is oriented towards an actual problem - management of the wastewater quality of a refinery “Lukoil” Bourgas. An important element of the strategy of the refinery was construction of artificial lakes (adaptation ponds) as a natural water treatment system, where an effluent water from the wastewater treatment plant has been stabilized. The ponds are shallow (average depth is 2 m) and cover an approximately 24 350 m2. This zone regulates the influence of effluent waters from the refinery on the Black Sea and plays an important ecological role. In the exploitation period (an approximately 20 years) the effort have been oriented to an intensification of the processes in the water system and an increase of the bioremediation efficiency. At this moment it is important to underline that there isn’t a suitable approach in bioindication and a scheme of the bioindicative relations, that can describe the mechanisms of the biodegradation in the sediments and the water body. The accent in this study is put on an investigation of microbiological characteristic of the pelagic ecosystem, because bacterioplankton are an integral functional and structural component of the plankton community. They comprise an important trophic level and play a key role in biodegradation and biodetoxification. The aim is to examine the abundance and the activity of indigenous micro-flora, and to assess the bioindicative potential of target biological indicators- microbiological and enzymological. The quantity and activity of the microbial plankton community with water chemistry in situ have been analyzed. This investigation is a part of whole hydrobiological assessment program that includes: 1) investigation of bioremediation activity in the sediments; 2) analysis of the quantity and quality of mineral oils and PAHs in the sediments (Krasteva A., D.Van Beneden, C.Van Keer, J.Topalova, D. Dimkov, 2001; C.Van Keer, A. Krasteva, D.Van Beneden, J.Topalova, R.Dimkov, D. Kozuharov, 2001); 3) investigation of structural and functional characteristics of zooplankton community (unpublished results).
MATERIALS AND METHODS The analysis were carried out with 10 water samples taken in November 2000 at 10 different sampling points situated in the way of water flow from 1-st to 4-th lakes (scheme 1). The reason was to assess the adaptation response of the microbial community to a different wastewater loading and different toxic pollutants.
Scheme 1 (C.Van Keer, A. Krasteva, D.Van Beneden, J.Topalova, R.Dimkov, K. Kozuharov, 1999) Samples were collected at 0,2 m below the water surface. Chemical and microbiological parameters were determined in situ. The samples for enzymological analyze was preserved at 40 C for 48 hours. Biological indicators - total bacterial count (cells/ml) (TBC) - heterotrophic bacterial count (cfu/ml) (HB) - phenol – degrading bacterial count (cfu/ml) (Ph.B) - dehydrogenase activity of plankton community (U/mg protein) (DHA) - coefficient K, that express ratio between total bacterial count and dehydrogenase activity of plankton community Chemical analysis The oxygen content (oxygen concentration (mg/l) and oxygen saturation (%)) and temperature (0C) were determined with “Hendylab OXIset”. The oxygen concentration was measured by Winkler’s (БДС,1989) method too. The chemical oxygen demand (COD) ( WTW-minitest, ISO 14 000), pH, total suspended solids (APHA,1982) and Sechii transparency (Tyler,1968) were also determined . Microbiological analysis The total bacterial count was determinated by the direct-count techniques (Наумова, 1999). The heterotrophic bacteria and phenol-degrading bacteria were enumerated with indirect “plate count” techniques according to the routine microbiological practices (Кузнецов,1989). The numbers of phenol-degrading bacteria are determined on a selective nutrient media – mineral content according to Furukava (1983) and phenol as a sole source of carbon and energy in a concentration of 100 mg/l. Enzymological analysis The DHA of plankton community was measured by reduction of tetrazolium salt –2,3,5 – triphenyl-tetrazolium chloride (TTC) to formazan (Звягинцев, 1980). The method was used in the following modifications: 25 ml water samples, incubation period was 24 hours at 28 0C, enzyme activity was terminated with acetic acid and extraction of the produced formazan was carried out with toluol. The absorption of the produced formazan was measured by specter-photometry at 487 nm. The protein content was determined by microbiuret method (Herbert, 1971). As a new element in the assessment of the DHA was used three different substrates of the enzyme activity: glucose, yeast extract and phenol. In that way has been simulated biodegradation of pollutants with a different chemical structure. The quantities of the above substrates were measured and compared by COD analysis. The COD of the artificial substrates were between 11 000 and 18 000 mg /l O2. The phenol was used as a model toxic pollutant for the assessment of the inhibition effect at two of the stations in the following concentration gradient : 0,2 mg/l, 0,4 mg/l, 0,6 mg/l and 0,8 mg/l. The phenol solution with concentration 0,8 mg/l had COD equal to COD of trivial substrate, and phenol solution with concentration 0,4 mg/l and 0,2 mg/l were with COD two and four times lower.
RESULTS AND DISSCUSION
Chemical characteristics
fig.1 Quantity of total suspended solids and chemical oxygen demand at different sampling stations.
fig.2 Water transparency at different sampling stations and mean depth of the ponds (1-9 th station). Transparency and measured depth at the 10-th station.
We calculated the oxygen deficit for different sampling points. The oxygen deficit was calculated as a difference between the saturation oxygen content at in situ measured temperature and in situ oxygen content at the same temperature. The results showd that oxygen deficit was significant and oxygen saturation was low (fig. 3). In the day (especially in the early afternoon) an appearance of the oxygen super saturation was typical (Wetzel, 1983). Such situation in adaptation ponds was not observed. The direct comparison between oxygen content in different sampling station is not done, because the diurnal oxygen variance in that kind of lakes was considerable.
fig.3 Oxygen concentration, oxygen saturation and oxygen deficit at different sampling stations
Microbiological and enyimological characteristics of indigenous micro-flora The total bacterial count was stable, relatively conservative and low sensitive parameter. Our investigation indicated, that the changes in the total bacterial count follows the COD changes in the way of water flow. This parameters decreased from 1-st to 4-th ponds (fig.1, fig.4a). fig.4 Total bacterial count and quantity of bacterial from the targeted physiological groups.
a) b) fig.4a Total bacterial count ( total bacterial count was determined with direct –count technique). fig.4b Quantity of heterotrophic bacteria (HB) and phenol-degrading bacteria (PhB) (target physiological groups were determined with indirect “plate-count” technique)
The abundance of the microorganisms from the targeted physiological groups varied in similar way. We observed a decrease in the quantity of two physiological groups (heterotrophic bacteria and phenol-degrading bacteria) from 1-st station to 4-th station, sharp increase of heterotrophic bacterial count for a 5-th station and extremely high quantity of phenol-degrading bacteria for 6-th station. Then these parameters decreased and remained low for the last four sampling station (fig. 4b). The changes in the indigenous micro-flora into the water body were the same like those found in the sediments (Krasteva A., Van Beneden D., Van Keer C., Topalova J., Dimkov D., 2001). That means, these parameters possessed an opportunity to control a key factor – contamination with aryl-containing hydrocarbons and other organic substances. For the description in details of the metabolic characteristics of the water community, was measured a dehydrogenase activity (DHA) of the plankton community, which is stimulated and inhibited from different categories of contaminants.
fig. 5 Changes of dehydrogenase activity of plankton community (DHA) and chemical oxygen demand (COD) at different sampling stations. The DHA was Measured toward two model substrates – glucose and yeast extract.
The DHA
was
stimulated by artificially added trivial
organic substrates in
different way
– the yeast extract increased
DHA from 130 to 2180 U/mg protein and the glucose stimulated DHA from 120 to
1990 U/mg protein. The DHA was inhibited 100 % from
а
phenol in concentration 0,8 mg/l. We used a lower concentration for two
sampling station (4-th and 7-th) situated in 2-th and 3-th pond. The DHA
measured toward glucose decreased five times
at
4-th station (410 U/mg protein) and four times at 7-th station (80 U/mg
protein) when the phenol was added in concentrations 0,2 mg/l. The decrease
was
ten times in 4-th station (160 U/mg protein) and 100 % in 7-th station when
phenol in concentration of 0,6 mg/l was added.
fig. 6 Relationship between the dehydrogenase activity and total bacterial count. This coefficient (proportion) expresses the relative activity of plankton community.
The relation between the DHA and total bacterial count has been doing parallel. This proportion expressed the relative activity of plankton community (fig. 6). The coefficient (K) gives the relation between measured activity and its structural biological base. The error in the coefficient is approximately 20 %, because a given percentage of the activity isn’t with microbiological origin. There was an interesting dependency between a bioremediation processes and COD loading – at high COD levels (680 – 530 mg/l O2) in 1-st and 2-th station, K was approximately 0,5 (bacterial count was high, but their activity is low). At lower COD levels (145-180 mg/l O2) the relative activity increased (K was 1-3), and at the lowest COD levels a relative activity decreased too but remain higher than the observed levels in the1-st and the 2-th station (fig.5, fig.6).
CONCLUSION A target bioindication, close to the naturale entity of the biotransformation processes was applied for the investigation of the bioremediation activity in the adaptation ponds. The accent was put on microbiological and enzymological parameters in combination with chemical water analysis in situ. A coefficient (K) was suggested that showed the relations between the bioremediation activity and Its structural biological base. The changes of this coefficient were followed in the process of water treatment. The next investigations will be oriented towards further decoding of the key mechanisms, that indicated and controld the bioremediation potential in the adaptation ponds.
ACKNOWLEDGEMENTS This investigation has been supported by The European Inco-Copernicus project IC-15- CT-98-0137. The authors are thankful for an assistance in the experimental work to E. Daskalova and G. Aleksieva.
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