By José Alfonso Álvarez González
Bioremediation is the process by which materials are added to the contaminated environment to be biologically remediated. This began to develop in the 1960s, and has been successfully applied in the treatment of soils contaminated with hydrocarbons since the 1980s and 1990s. It is characterized as a low-cost and environmentally safe technique.
From the Ingenito Ecological Reserve area, located in Havana Bay, samples of coastal soil contaminated with petrolized hydrocarbons are taken with a view to carrying out Biotreatment experiments, by the method of Biostimulation, at laboratory scale, simulating the conditions treatment in the field. Using a factorial system of 23, it is determined which variants (hydrocarbon concentration, presence or absence of bagasse, and presence or absence of nutrients) provide a higher rate of biodegradation for subsequent field application. Monitoring of the native bacterial population of the soil and the production of CO2 are carried out. At the same time, the rate of biodegradation over time is determined. After the 90 days of the experiment, the greatest decrease in hydrocarbons is obtained when bagasse and nutrients are used separately for both concentrations of hydrocarbons tested.
Bioremediation is the process by which materials are added to the contaminated environment to be biologically remediated (Head, 1998). It is characterized by being a low-cost and environmentally safe technique.
This technique can be applied in-situ or ex-situ depending on the place where the treatment is carried out, applying the biostimulation method, which is based on adding nutrients to stimulate the growth of autochthonous microorganisms, or by the method of bioaugmentation by which exogenous microorganisms with the ability to degrade hydrocarbons are added. Either by one method or another, the biodegradation process is greater as a consequence of better control / effectiveness of abiotic factors such as temperature, pH, aeration, mixing, humidity; and biotics as activity and growth of the microorganisms that affect the process (Huddleston and Bleckmann, 1986; Infante and Arias, 1993; Ercoli, 2001)
It is important to note that the economic factor plays an important role when selecting the method to use, as well as the desired speed for cleaning the site. For this reason, before carrying out the treatment on a large scale, a prior study of these factors is required at the laboratory level for subsequent application in the field.
The objective of this work is to determine, on a laboratory scale, the variant that provides the highest biodegradation rate, with a view to bioremediating soils contaminated with hydrocarbons.
Materials and methods
Samples of coastal soil contaminated with hydrocarbons were taken at different points in the area of the Ingenito Ecological Reserve, located in the Bay of Havana. The samples were taken in the shape of a star and all were subsequently mixed to guarantee greater homogenization and representativeness. For the initial determination of the chemical analyzes, a fraction of the composite sample was collected and packed in nylon bags, preserving in freezing until processing. At the same time another fraction of the sample was separated for microbiological analysis. The rest was used to set up the experiments.
For the microbiological analysis, 10 g of soil were taken and dissolved in 100 ml of saline solution with Tween 80 to achieve the dispersion of the hydrocarbon from the soil particles. After this mixture was shaken, serial dilutions were made and a portion of it was inoculated on nutrient agar medium. After 24 hours of incubation, they were read (ISO 4833: 1991).
CO2 production (Respirometry)
The measurement of the CO2 produced per unit of time in a given area is an indirect measure of the biodegradation process since it aims to evaluate the respiratory activity of soil microorganisms during the degradation process of organic compounds. For this, a plastic container containing KOH (0.1 N) was placed covered by another larger container in such a way as to avoid any exchange with the outside environment. These remain for 18 hours, during which time the CO2 released by the biological activity is adsorbed by KOH, this is titrated with HCl solution (0.1 N) and The difference between this assessment and that obtained from a blank, gives us the milligrams of CO2 produced per m2 per hour (Viale and Infante, 1997).
The samples were homogenized, dried in the oven and sieved through a sieve with a pore diameter of 2 mm. From here a representative sample was taken to be analyzed. The determination of fats and oils was carried out by the method of Abboud S.A, 2000 and the determination of total Hydrocarbons by the APHA 5520F method (APHA, 1991; APHA, 1995).
To select the most feasible variant to be used in a pilot scale, a semiquantitative full factorial design 23 was performed, where as independent variants the following were used:
|X1:||Hydrocarbon Concentration (1% and 3%)|
|X2:||Presence - Absence of Bagasse|
|X3:||Presence - Absence of Nutrients|
The composition of each of the experiments is shown in Table 1. In all cases the final mass was 2000 g and the amount of Urea and DAP was determined according to the C / N = 60 and C / P = 800 ratio. The experiments (See Annex) were manually aerated every 2 or 3 days and periodically moistened to maintain a relative humidity of approximately 80%. Monthly samplings were carried out for the determination of fats and oils and total hydrocarbons and for the determination of CO2 and bacteriological count.
Table 1. Composition of the Experiments.
|Experiment||1% HC (g)||3% HC (g)||Clean soil (g)||Nutrients (g)||Bagasse (g)|
DAP: diaminophosphate (fertilizer)
The whole experiment was carried out with 3 repetitions. After the analysis of similarity of variance by the Bartlett method (Lerch, 1977), the triple classification analysis of variance and Duncan's multiple range comparison test (Lerch, 1977) were performed, in which the means that did not they differ and are expressed with the same letter. In all tables and graphs the means are reported with the confidence interval for a significance coefficient of 5%. The results were processed with the statistical package STATGRAPHICS Plus.
Results and Discussion
Behavior of Microbial Populations
Table 2 shows the monthly results of the bacterial concentrations during the course of the experiment. In all cases, the concentration ranges between 106 and 107 mostly, acceptable levels for the development of biodegradation processes. From the values reported in the table and the distribution obtained graphically (Figure 1) there is evidence of a decrease in the concentration at 28 days, an expected result considering that when mixing clean soil with contaminated soil, part of the initial biota present on clean soil it may not adapt to the new conditions imposed.
Table 2. Colony Forming Units (CFU) per gram of soil.
|Experiment||t = 0 days||t = 28 days||t = 60 days||t = 90 days|
In the particular case of experiment 3, 4, 7 and 8, the highest initial values are observed (0 - 60 days). This could be justified by the presence of bagasse, which also contributes microorganisms and allows greater oxygenation of the soil by improving its texture. Furthermore, the presence of nutrients also favors this behavior for experiments 7 and 8. From 60 to 90, the values of the bacterial concentration are lower and remain stable up to 90 days (Figure 1).
Figure 1. Behavior of Bacterial Populations.
The respirometric values are shown in Figure 2, where it can be seen that in all the experiments an initial decrease in CO2 production is observed, this is corroborated with the values of Colony Forming Units obtained in Figure 1 and is due to the adaptation of autochthonous microorganisms to the new imposed environment. However, in all cases typical patterns of biodegrative processes are observed, which tells us that oil degradation is taking place (Infante, 2001).
Figure 2. CO2 production.
Table 3 reports the concentration levels of total oil hydrocarbons obtained at different times, observing that already 30 days after the experiment started, acceptable degradation values are obtained (greater than 25%) (Infante, C, 1999 ). This guarantees that the biodegradation process carried out is effective and that soil sanitation by the Biostimulation technique is viable for this type of pollutant and under these conditions (Infante, C; 1999).
The highest hydrocarbon degradation rates after 90 days are obtained in experiments 3 and 4 (with bagasse) and 5 and 6 (with nutrients), for 1 and 3% hydrocarbon concentration respectively. However, this does not completely coincide with the results obtained from the microorganism count, where experiments 5 and 6 had the lowest bacteriological concentrations. This means that the high values in the colony forming units, for the other experiments, were due to the presence of bagasse heterotrophic bacteria and not necessarily to degrading bacteria.
Table 3. Total Hydrocarbon Concentration, expressed in%
|Experiment||t = 0 days||t = 30 days||Degradation rate 30 days (%)||t = 90 days||Degradation rate 90 days (%)|
It is good to point out that the quantitative determination of hydrocarbons in soil is complex since most of the techniques are based on the extraction of various fractions by solvents. The solubility of these fractions in different solvents is variable. Hence, depending on the method used for the determination of hydrocarbons, different values will be obtained, which for certain types of soils and hydrocarbons can be very marked (Ercoli, 1999). On the other hand, it is difficult to take a sample when it comes to heavy crude oil on the ground and for these reasons, in some cases, higher values of total hydrocarbons are obtained at 90 days than at 30 days. However, the values are always less than those obtained at time zero.
Table 5 analyzes the results 90 days after starting the experiment.
Table 5. Differences in Degradation Rates at 90 days
|Experiment||Degradation rate 90 days (%)|
If the results obtained between variables 3, 4, 5 and 6 are taken into account, there are no significant differences from the statistical point of view for a 95% reliability of the values. The differences are given for variants 1 and 2, where there are no nutrients or bagasse to facilitate biodegradation and for variants 7 and 8 because non-degrading heterotrophs are favored by the presence of both.
For variants 3 and 4, the values are higher due to the contribution of the bagasse microorganisms and because soil aeration is favored and for variants 5 and 6, due to the presence of nutrients that stimulate the autochthonous degrading microorganisms.
However, in a general sense, any variant could be applied since satisfactory results are produced in the 8 experiments, so the best variant from an economic point of view would be 1 and 2, where only clean soil is mixed with contaminated soil.
- The statistical analysis applied to the experiments using impacted soil from the Ingenito area showed that there are no significant differences between the applied variants.
- The highest biodegradation rates are obtained when conditioning material and nutrients are used separately for both hydrocarbon concentrations.
- The biotreatment process is effective for this type of soil and pollutant.
The coastal soil of the area of the ecological reserve of the Bay of Havana can be recovered by mixing it with clean soil and bagasse or nutrients separately, for greater removal of hydrocarbons. The use of one or the other depends on economic conditions. However, sanitation simply aerating the soil with agricultural equipment is not ruled out. In this case, the same oil removal values would be obtained but in a longer time.
* Lic Esther Ramos Padrón, MSc José Alfonso Álvarez González, Lic Ana Núñez Clemente, Tec. Gisela Novoa Rodríguez, Lic. Sandra Miller Palmer
Petroleum Research Center
Washington # 169, Cerro, Havana, Cuba
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