Chen Yan-jun, Wang Hong-qi, Zhang Ping, Yunying

State Key Joint Laboratory of Environmental Simulation and Pollution Control, College of Water Sciences, Beijing Normal University, Beijing 100875

Abstract: Petroleum hydrocarbons cause serious environmental problems. However, the mechanism of degradation by microorganism was partially unknown. Localization of degradation enzyme in two bacteria for n-Hexadecane, production and characteristics of n-Hexadecane-degrading enzyme were studied in this paper. The cellular extracts of l bacterium were fractionated into extracellular, periplasmic and cytoplasmic fractions after osmotic shock. The results showed that the main enzyme, in the 1 bacterium, which are cytoplasmic and membrane-associated enzymes, decomposed mainly n-Hexadecane. And most of the enzyme activities (both 100%) in 2 bacterium were detected in the cytoplasmic and extracellular extracts. Moreover, culture conditions have a significant effect on the enzyme activity produced in hexadecane cultures of 1 bacterium and 2 bacterium, and the optimum of the concentrations of hexadecane is 10mg/L.The rhamnolipid(2mM) was the most effective in stimulating producing the main enzyme of degradation for n-Hexadecane. The enzyme is active when the pH value is in the range of 7.0~8.0, and the most suitable pH value is near 7.0. The enzyme is active at temperatures up to 30℃, but it is instable at higher temperatures.

Keywords: microorganism; enzymatic degradation; n-Hexadecane; osmotic shock

Manufacture, transportation and utilization of petroleum hydrocarbons have brought about frequent occurrences of soil contamination with petroleum. Bioremediation is an innovative technology that depends on the indigenous soil microorganisms to transform or mineralize the organic contaminants. However, because the components of the oil and the processes of the transformation, metabolism and degradation are quite complicated, the mechanism of biodegradation by microorganism was not entirely known. Many enzymes are in either extra-cellular, periplasmic or intra-cellular and may be concerned with the processing of transported contaminants (Kohji Miyazaki, 1997). Therefore, the localization of enzymes is a key step in the degradation of compounds, and localization of many enzymes has been reported, e.g. Zhang Chendong reported the degradation an aromatic sulfur-containing compound mainly locates in cell and on the membrane (Zhang Chendong, 2000). Di-n-butyl phthalate-degrading enzyme was an intra-cellular enzyme (Zeng Feng, 2000). And yet there is little or no information available about the distribution of enzyme in bacteria which can degrade petroleum hydrocarbon. Here we used n-Hexadecane as representative pollutant, and the microorganism was taken from Daqing oil field. Experiments were done in the laboratory to deal with localization of the degradation enzyme in two bacteria for n-Hexadecane. Otherwise, the production and characteristics of n-Hexadecane-degrading enzyme were determined.

Materials and Methods

Chemicals. Hexane(99.9% pure) was obtained from J&K Chemicals LTD. n-Hexadecane(99% pure) was obtained from Germany. Magnesium sulfate(analysis pure) , Ammonium chloride(analysis pure), Sodium nitrate(analysis pure), Sucrose(analysis pure), D-Glucose(analysis pure), di-Potassium hydrogen phosphate(analysis pure) and  Tris(99.5%  pure)were obtained from the Beijing Chemical Factory.

Bacterial cultures. Two wild-type strains are capable of metabolizing n-Hexadecane, which were isolated from soils polluted by petroleum. The mineral salts medium (MSM) containing 0.4%Na₂HPO₄, 0.15%KH₂PO₄, 0.1%NH₄Cl, 0.02%MgSO₄ 7H₂O, 0.0005% iron ammonium citrate, 0.0015%CaCl₂, and 1ml of trace elements solution per liter. Cultures were incubated in duplicate in 250ml flasks containing 100ml MSM. Glucose and hexadecane were used individually as sole source of carbon and energy. Flasks were inoculated with 10% inoculums of 1 bacterium, 2 bacterium individually and incubated on a rotary shaker (150rpm) at 30℃。

Biodegradation studies.

Growth studies. Initial studies to examine biodegradation of n-Hexadecane by 1 bacterium and 2 bacterium were conducted by measuring the bacterial growth in medium under agitation at 150rpm.Inoculations were made in MSM supplemented with 0.01% n-Hexadecane and Glucose individually. Samples were removed at regular intervals and measured by optical density (OD) measurement at 600nm.

Determination of n-Hexadecane mineralization.  Hexadecane consumption by 1 bacterium and 2 bacterium was determined indirectly by extracting residual n-Hexadecane in the solvent wash obtained from the accumulation assay. Briefly, bacteria were grown to mid-growth phase in flasks containing n-Hexadecane and harvested by washing with the hexane. The resulting solvent washes were collected, pooled, and used for measuring extra-cellular n-Hexadecane.  Hexadecane was extracted twice with 10ml of hexane. The organic phases were collected, dehydrated over anhydrous sodium sulfate, concentrated, and used for GC analysis of n-Hexadecane. Consumption of n-Hexadecane was defined as the difference between the initial mass of n-Hexadecane added to the cultures and the remaining total mass after incubation, both intra- and extra-cellular. The extraction efficiency of n-Hexadecane, determined from the recovery of the internal standard from the sample, was above 80%.

Gas Chromatographic analysis. Hexadecane was quantified by GC using a Varian5.5 gas chromatograph equipped with a flame ionization detector (FID) and HP-SE-54 capillary column (30m). Nitrogen was the carrier gas at a flow rate of 16～20cm/s. Samples of 1μl were injected via a split injection port at a split ratio of 60：1. The injection port and detector temperatures were constant at 250 and 300℃, respectively. The column temperature was kept initially at 100℃ for 2 min, then increased at 10℃/min and finally held for 10 min at 250℃,. The mass of n-Hexadecane accumulated in cells was calculated using calibration curves with individual authentic standards, normalizing to the internal standard.

Localization of degradation enzyme.

Osmotic shock method. Recovery of enzyme was carried out immediately after sampling by the  cold osmotic shock method (Park and Lee,1998) as follows. Cells from 20ml culture were harvested by centrifugation at 6000rpm for 10min at 4℃. The pellet was washed with cold distilled water twice. Then the cell pellet was re-suspended in 10mM Tris-HCl (pH8.0), followed by centrifugation at 6000rpm for 10min at4℃.The pellet was re-suspended in 25%(w/v) sucrose . After incubation with gentle shaking at room temperature for 10 min, cells were harvested by centrifugation for 10 min at 10000rpm and 4℃, and enzyme was collected. The cell pellet was re-suspended in cold distilled water and was incubated for 10 min in an ice-water bath with vigorous shaking. After the cell pellet and supernatant were separated by centrifugation for 10 min at 13000g and 4℃, the supernatant was recovered as the periplasmic fraction. The pellet was re-suspended in 10mM Tris-HCl (pH7.5), and the solution was sonicated thoroughly to disrupt cells. The inclusion bodies from the sample were isolated by centrifugation at 15000rpm for 20min at 4℃. The supernatant was recovered as the soluble protein fraction.

Measurement of enzyme activites. To determine the enzyme activity from crude extract, the reaction mixture contained 2ml extra-cellular extract, periplasmic extract, and cytoplasmic extract individually, 8ml of 0.001% n-Hexadecane in 50mM Tris-HCl (pH7.2).The solution was incubated at 30℃ for certain time with continuous shaking. Then n-Hexadecane was extracted twice with 10ml of hexane. The organic phases were collected, dehydrated over anhydrous sodium sulfate, concentrated, and used for GC analysis of n-Hexadecane.

Optimization of the enzyme production. A large number of enzyme assay procedures have been developed which differ not only in certain conditions of the assay, e.g. rhamnolipid concentration or the carbon source employed, but in the underlying principle of the quantification of the enzyme activity.

Characterization of crude enzyme. The reaction was carried out at various temperatures ranging from 15℃ to 40℃ and the enzyme activity at different temperature points were measured in order to find out the temperature optimum of enzyme. Similarly, the enzyme assay was carried out at different pH levels from 6.0 to 9.0 using 50mM Tris-HCl buffer.

Results and Discussion

Biodegradtion tests.

As observed in Fig.1, 1 bacterium and 2 bacterium grown in glucose exhibited stationary growth (as monitored by OD₆₀₀) over the first 21h. A similar set of experiments were performed with n-Hexadecane as a substrate. The relative growth of bacteria on 10mg/L n-Hexadecane showed a slow, steady increase over a period of 158h(Fig.2, Fig.3). Cell growth entered in the stationary phase after 96h and 47h incubations for 1 and2 bacterium. Thus it is to be noted that both strains utilized n-Hexadecane slowly, while the growth rate of 1 and 2 bacterium in glucose were more rapid. Due to n-Hexadecane extremely hydrophobic nature, it partitions readily into liquid organic phases and exists in aqueous phase only at limited concentrations. This makes uptake of n-Hexadecane by bacteria cells difficult. In bacterial cultures where glucose was dissolved in aqueous solution is utilized easily. The data in Fig.2 and Fig.3 suggest that the rate of two strains growth on 100mg/L n-Hexadecane increased rapidly during the early stages of the experiment (11h), however cells dropped significantly to low levels after 11h. The decreased growth of both strains with increasing n-Hexadecane concentration was consistent with an increasing metabolic load for degradation of the organism. In addition, this is often due to accumulation of the hydrophobic compound in bacterial membranes which can cause devastating effects on membrane structure (Sikkema J, 1994; Sikkema J, 1995 ).

As illustrated in Fig.4, approximately 99% of the 10mg/L initial concentration of n-Hexadecane was degraded within 7d. 1 bacterium and 2 bacterium could take in and decompose 59% and 69% of the originally added 100mg/L n-Hexadecane during this time, respectively. This shows the degradation

capacity of two strains were better. For hexadecane, biodegradation was enhanced at the lower n-Hexadecane concentration (10mg/L). The pattern of n-Hexadecane biodegradation was similar to that of bacterial growth.

Localization of degradation enzyme.

The cellular extracts of bacteria were fractionated into extra-cellular, periplasmic and cytoplasmic fractions after osmotic shock. As illustrated in figure5, the cytoplasmic extract of 1 bacterium is capable of degrading 100% of the n-Hexadecane in 24h. On the other hand, the n-Hexadecane consumption of the membrane-associated and extra-cellular extract were 86% and 47%, respectively. It was revealed that some enzyme proteins residing in the periplasmic space have important functions in the detection and processing of essential nutrients and their transport into the cell (Yuji Nagta, 1999). Therefore, in the 1 bacterium , the main enzymes, which exist in cytoplasmic and membrane-associated extracts, decomposed mainly n-Hexadecane. The enzyme activities of each fraction in 2 bacterium were measured as described previously; most of the enzyme activities (both 100%, Fig.5) were detected in the cytoplasmic and extra-cellular extracts. The enzymes were not detected in the periplasmic space (Fig.5), indicating that enzymes cross both cell membranes without stopping in the periplasm. This result suggests that enzymes are in the periphery of the cells and in the cytoplasmic space.

In contrast, glucose experiments showed that n-Hexadecane was not completely utilized by either extra-cellular, membrane-associated or intra-cellular extracts within the 24h time frame of the experiment (Fig.6). Differences in growth conditions may affect the distribution of enzymes, since we found the lower enzyme activity in glucose-grown cells compared with cell grown on n-Hexadecane.

Optimization of the enzyme production.

As illustrated in figure7, when both strains grown with hexadecane(10mg/L) as sole source of carbon and energy, extra-cellular, membrane-associated and intra-cellular extracts are capable of degrading 100% of the n-Hexadecane present in less than 24h. The n-Hexadecane consumption of the membrane-associated fraction was less than 85%, when both strains were grown under a rotary shaker with 1mg/L hexadecane as a carbon source. Moreover, the extra-cellular and intra-cellular enzyme activity was not reduced, and the specific

activity of the biodegradation was very high. The enzyme activity was significantly reduced with the higher concentration of hexadecane(100mg/L). As illustrated in figure7, approximately 12.8% and 43% of the n-Hexadecane respectively, were degraded by intracellular extracts of both strains within a period of 24h. We conclude that high concentrations of hexadecane cannot promote the release of in-membrane enzyme by both strains of bacteria, since the growth of the bacteria seemed to be limited. Therefore, the results of this study suggest that culture conditions have a significant effect on the enzyme activity produced in hexadecane cultures of both strains, and the optimum of the concentrations of hexadecane is 10mg/L.

Figure 8 showed that the production of the main enzyme was enhanced at the low rhamnolipid concentration. For 1 bacterium and 2 bacterium, the rhamnolipid (2mM) was the most effective in stimulating the rate of biodegradation for n-Hexadecane. The results were similar to the experiment done by Itoh（Itoh,1971）.However, for n-Hexadecane, the rate of mineralization decreased for both strains in the present ofrhamnolipid(>2mM). Zhang and Miller found that low levels of added biosurfactant could enhance the biodegradation process. This is because, the addition of rhamnolipid have much impact on the growth of either strain (Ragher A, 2000).

Characteristics of n-Hexadecane-degrading enzyme

Effect of pH on the activities of enzyme. The activities of enzyme was quite sensitive to pH(Fig9). The activity of the enzyme was highest between pH 7.0 and pH 7.5. As the pH was increased above 7.5, there was a slight decrease in activity that resulted in a decrease in the degradation rate. On the other hand, as the pH was decreased from 7.0 to 6.0, activity decreased significantly, resulting in an decrease in activity from 100% to <60%.

Effect of temperature on the activities of enzyme. The activity of enzyme was dependent on   the temperature during incubation. The activity increases with temperature of incubation until approximately 30℃ and then the activity of enzyme significantly decreased below 35℃.

Thus, the enzyme is active when the pH value is in the range of 7.0~8.0, and the most suitable pH value is near 7.0. The enzyme is active at temperatures up to 30℃, but it is unstable at higher temperatures.

Conclusions

This study clearly demonstrated that the main enzymes in the 1 bacterium, which are cytoplasmic and membrane-associated enzymes, decomposed mainly n-Hexadecane. And most of the enzyme activities (both 100%) in 2 bacterium were detected in the cytoplasmic and extra-cellular extracts. Moreover, culture conditions have a significant effect on the enzyme activity produced in hexadecane cultures of 1 bacterium and 2 bacterium, and the optimum of the concentrations of hexadecane is 10mg/L.The rhamnolipid (2mM) was the most effective in stimulating producing the main enzyme of degradation for n-Hexadecane. The findings of this study suggest that the most suitable pH value is near 7.0, and the enzyme is active at temperatures up to 30℃.

Acknowledgments

We thank LIANG Sheng-kang for providing rhamnolipid for assistance in part of experiment work. We would like to thank Xu Jing and her husband for their assistance with revising this paper.

This work was supported by National Natural Science Foundation of China （40472129）.

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