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Trends in Bioremediation and Phytoremediation, 2010: 429-451 ISBN: 978-81-308-0424-8 Editors: Grayna Paza

25. Hydrocarbon bioremediation and phytoremediation in tropical soils: Venezuelan study case
1

Infante, C.1, Morales, F.2, Ehrmann, E. U.2, Hernndez-Valencia, I.3 and Leon, N.4

Instituto de Ciencias de la Tierra, Facultad de Ciencias, Universidad Central de Venezuela; 2Departamento de Procesos y Sistemas, Universidad Simn Bolvar, Venezuela; 3Instituto de Zoologa y Ecologa Tropical Facultad de Ciencias, Universidad Central de Venezuela; 4COPRESA. Consultora Ambiental

Abstract. Bioremediation has been widely used in tropical oil producing countries to remediate oil contaminated soils, crude oil pit bottoms and to treat oily wastes like drilled cuttings and oily tank bottoms. This paper presents a brief review of some of the most relevant research and field bioremediation studies in Venezuela. In agreement with international publications, lab results as well as field experience show that for open bioremediation processes, like landfarming and composting, biostimulation of indigenous microbial population is always more effective than bioaugmentation. Moreover, the success of any open bioremediation process depends largely on the physical and chemical conditioning of the media or soil. This conditioning is accomplished maintaining - proper humidity, aeration, addition of organic conditioners, like detritus or manure, and nutrients to adjust the carbon/nitrogen and carbon/phosphorus ratios, among other key factors. Similarly to results from other countries, experimental findings in Venezuela show that crude oil and oil products are not 100% biodegradable. Oil properties like API gravity, the content of aromatic and polar fractions, H/C ratio, distillation residue, among others, determine oil biodegradability. For instance, low API gravity crude oils show negligible biodegradability. Consequently, bioremediation is not a sound choice to treat heavy crude oil contaminated soils or heavy crude oil drilled cuttings. Bioremediation removes the more toxic and lower molecular weight hydrocarbons. The remaining recalcitrant TPH (Total petroleum hydrocarbon) fractions are much less or not toxic at all; thus, they do not pose significant environmental and health risk. However, these residual TPH fractions arise discussions regarding the compliance
Correspondence/Reprint request: Dr. Infante, C., Instituto de Ciencias de la Tierra, Facultad de Ciencias, Universidad Central de Venezuela. E-mail: carmeninfante66@gmail.com

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of local cleanup criteria and/or the allowable TPH levels in soils after completion of the bioremediation process. The use of bioremediation to meet Venezuelan, Colombian and Ecuadorian soil TPH limits without dilution of the recalcitrant TPH fractions is discussed. This paper also presents a discussion about the technical basis of the regulatory limits for TPH in soils in these three South American oil producing countries. Finally, lab scale and greenhouse results of phytoremediation studies are presented. The research in this promising field is focused on the screening and identification of plant species that could have phytoremediation potential for waste treatment. Oil effects on seed germination, biomass production and leaching to groundwater, as well as the ability of species to reduce TPH in soils, are discussed. Future research to estimate mass balances and the fate of hydrocarbons in the soil-plant system is still needed.

Introduction
Venezuela is an important oil producing country since the early 20s. Since then, intense E&P efforts have been developed in the country which resulted in significant discoveries and continual production. For instance, heavy crude oil reserves at the Orinoco Oil Belt are among the largest worldwide. E&P activities as well as transport and refining operations generate large amounts of oily wastes like drilled cuttings, spill contaminated soils and tank bottom sludge. Historically, these wastes used to be confined in unlined pits; however, nowadays they need to be treated to comply with legal regulations. For this purpose bioremediation has deserved special attention due to its simplicity, low cost, low environmental impact and public acceptability. Additionally, tropical conditions stimulate microbial activity and allow the application of bioremediation throughout the year. Not surprisingly, to date bioremediation is the most widely used technique to clean up oil contaminated soils in Venezuela. Nevertheless, its application is limited by crude oil biodegradability which depends on the oils properties. Research and application of this technique to remediate oil contaminated soils began in Venezuela in the 1990s. Main efforts were focused on the stimulation of microbial activity and improvement of soil properties. Since most of Venezuelan reserves correspond to heavy crude oil, recent research efforts are directed towards the treatment of soils impacted with these oils. Additionally, alternate biological processes such as phytoremediation are currently being evaluated to treat recalcitrant crude oil fractions. This paper compiles some of the main findings on bioremediation studies in Venezuela; results thereof could be applicable to other tropical regions.

Biodegradation and bioremediation

Biodegradation is the partial simplification (transformation) or complete destruction (mineralization) of the molecular structure of environmental pollutants through physiological reactions catalyzed by several microorganisms [1, 2, 3]. Bioremediation is the intentional use of a biodegradation processes to eliminate environmental contaminants from sites where they have been released either intentionally or inadvertently. Bioremediation technologies use the physiological potential of microorganisms to eliminate or reduce the concentration of environmental contaminants to levels that are acceptable to site owners and regulatory agencies that may be involved [2, 3]. In an ecosystem, biodegradation is the natural organic matter decomposition and carbon loss process through microbial action [4] whereas the term bioremediation

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specifically applies to the remediation of contaminated soil. The term was coined in the 80s, particularly due to the large rise and development of the technique as a result of the Exxon Valdez oil spill on the Alaskan shoreline [5]. Conversely, the term hydrocarbon biodegradation has been widely used since the 60s due to the fact that there was abundant information regarding the capabilities of a wide variety of microorganisms to metabolize hydrocarbons [6].

Biostimulation vs bioaugmentation
Two main strategies are used for bioremediation; that is, bioaugmentation and biostimulation. Bioaugmentation refers to the use or application of biological preparations (inoculums,) grown in a laboratory and added in large quantities to the field. The microorganism activity is enhanced through addition of suitable bulking agents as well as N and P in order to adjust the C/N and C/P ratios. Throughout the process, appropriate aeration and moisture control must be assured [3]. The inoculums can be either specific bacteria or fungi strains, or mixtures thereof. Both, fungi and bacteria, play important roles in hydrocarbon biodegradation [6] none of them can be regarded as more relevant than the other. Nevertheless, there are significant differences between the fungi and bacteria hydrocarbon metabolisms [7, 8]. Relative dominance of one species over the other varies with the ecological soil conditions [9]. In biostimulation approaches no microorganism preparations are added. However, similar soil conditioning techniques are applied; that is, nutrient addition, aeration, moisture and bulking agent control, to stimulate the indigenous micro flora in the soil or in the crude oil waste [3]. The dispute regarding the more effective bioremediation strategy still persists. For instance, it has been reported that in the tropics a higher diversity of microorganisms able to readily degrade hydrocarbon compounds is ubiquitously present in the soil. Among other reasons, the steady and warm temperatures throughout the year in tropical regions may account for this fact. Since high humidity and solar radiation also favor biostimulation, this is the most commonly used option in tropical areas like Venezuela [10]. In addition to the extra cost related to the inoculation of microorganisms, there are several reasons why bioaugmentation is not the best choice. First of all, it is difficult to cultivate a representative population of bacteria and fungi capable of degrading hydrocarbons because it has been found that just over 1% of total microorganism population present in soil can grow under laboratory conditions. It is virtually impossible to reproduce the physiology and metabolic interactions among populations of bacterial species that take place in a natural soil or a crude oil contaminated waste. Microorganisms, as every community on Earth, interact from an ecological point of view and are regulated by the microclimatic conditions of each soil, which cannot be recreated in the laboratory. Even though molecular biology techniques have allowed major advances in the understanding of microbial communities, there is still a long way to go [11]. Consequently, some species that may be dominant in a natural community might not grow under laboratory conditions; thus, the interactions present among the communities in the original substrate are precluded in the laboratory culture [12]. These interactions may be relevant for the degradation process and their absence may compromise the success of the remediation. Additionally, the variety of compounds present in petroleum, comprising molecules with aromatic, aliphatic and heteroatomic moieties can only be degraded by the concerted action of different microbial communities. As a result of all the above mentioned factors, commercially available microbial consortia for field applications turn out to be little effective for the remediation of

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crude oil contaminated soils. Moreover, it has been reported that, because of predation and competition with the indigenous microorganisms, the introduced cultures will disappear faster; therefore, continuous addition of the foreign consortia is mandatory for successful clean-up [13, 14]. Experience in Venezuela has shown that biostimulation is a better option than bioaugmentation. For instance, table 1 compares bioaugmentation vs bioestimulation results from a bioremediation field trial of an oily mud from a western Venezuelan oil pit [15]. For this study, an inoculum was chosen among bacteria isolated from three different sources; that is, petroleum contaminated soil, non-contaminated soil and dry sludge from a sewage treatment plant. The selection was based on laboratory biodegradation assay results. Those assays revealed the sewage treatment plant sludge bacteria as the most effective for the removal of hydrocarbons from the contaminated test soil. For field evaluation 4 m2 plots with 10% w/w crude/soil ratios of the same oily sludge used for the laboratory tests were prepared. For scaling purposes, the inoculums were applied at 108 CFU/gsoil doses. The plots were fertilized with urea and triple superphosphate to yield a C/N ratio of 60 and a C/P ratio of 800; moisture was maintained at 60% of the field capacity and, for aeration, the soil was thoroughly mixed three times a week with the help of a rake. For each treatment, three replicates were prepared; the test was carried out over a six month time period. Field results showed that the addition of the inoculums did not significantly enhance the biodegradation rate (p <0001) as compared to the pure biostimulaton treatment. Thus, biostimulation was considered more effective than bioaugmentation for bioremediation purposes, given the extra costs due to the inoculum addition that did not yield any benefit. These results are consistent with other studies, where the addition of laboratory grown inoculums or commercial microorganism preparations were effective only at laboratory scale, but in very few cases, at field scale [6, 16-19].
Table 1. Biodegradation rate, as a percentage of hydrocarbon removal (saturates plus aromatics) during biostimulation and bioaugmentation tests [15]. Treatment 0 60 Sampling Time (days) 90 150 180

Biodegradation (%) Soil + Oily sludge +A +F Soil +Oily sludge +A +F+I 0 0 60 68 72 71 81 75 80 78

A= Aeration F= Fertilizers I= Inoculum (Sewage sludge from a water treatment plant)

Factors affecting bioremediation processes

Bioremediation is not a complex process; nevertheless, it requires careful optimization and control of several interplaying factors. Among these factors, soil type, soil moisture, temperature, oxygen and nutrient availability, as well as the use of bulking agents, play key roles. Even though the importance of these aspects upon bioremediation has been recognized, only few publications refer to the influence of these parameters under tropical conditions [11]. It is known that crude oil contaminated soils have limited nutrients for the bioremediation to proceed at a reasonable rate. The hydrocarbon represents an

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excessively large carbon source; thus, relative nitrogen and phosphorus availability is too short for the microorganism to thrive, and become the limiting nutrients [20, 21]. Consequently, the soil must be amended with adequate nitrogen and phosphorus sources at proper concentrations, since an excess or a deficiency thereof will inhibit the microbial activity [10, 22]. Interestingly, this nutritional condition may selectively affect the biodegradation rate of specific hydrocarbon families present in petroleum, such as the saturated fractions for instance [23]. C/ N ratios of 10:1 [24,] 20:1 [25,] 50:1 [26,] 60:1 [27] and even 560:1 [28,] have been reported. C /P ratios from 100:1 [29, 30] up to 800:1 [27] have been recommended. Trials performed in Venezuela have shown that a C/N ratio of 60:1 together with a C/P ratio of 800:1 are appropriate nutritional conditions for bioremediation purposes. These proportions have been widely used for clean-up of sludges from crude oil waste pits [15, 31] soils contaminated with different types of crude oil [32] as well as oil based drilled cuttings [15]. Organic conditioners such as rice husk, wood chips, spent compost, sugar cane bagasse, litter from tropical trees or grass, poultry manure, etc. play an important role in improving the structure and porosity of the contaminated soil. Some of these amendments also could be an extra source of nutrients and microorganisms [33-36]. These bulking agents are essential for bioremediation to proceed, particularly in soils that exhibit poor structure, high infiltration and low moisture retention capacity, such as sandy soils. Similarly, loamy soils, characterized by a high moisture retention capacity, but very poor oxygen diffusion rates do not favor bioremediation processes. These conditions too might be overcome with the use of appropriate bulking agents. Comparison of the biodegradation rate of an oil based drilling mud mixed with clay loamy soil and silty clay soil showed better performance with the first soil. In silty clay soils, biodegradation is slower, due to hydrocarbon sorption on clay particles that reduce the bioavailability of the pollutant to the microorganisms [37]. Table 2 shows results of the biodegradation % of oil based drilled cuttings from western Venezuela, where two types of bulking agents where mixed together with a sandy loamy soil. Data shows that with either of the two conditioners tested, the biodegradation rate was significantly higher as compared to the control (without conditioner). These results confirm the important role played by bulking agents to improve the structure and porosity of organic wastes and thus, promote the biodegradation process [15]. Temperature is another factor that affects the biodegradation rate. At higher temperatures, oil viscosity decreases and the diffusion rates and spreading of the organic compounds increase. This phenomenon results in a larger pollutant water interface which enhances the pollutant microorganism contact and thus, the oil degrading microorganism colonization. However, the same physical phenomenon can lead to greater hydrocarbon absorption on the vegetable biomass or organic matter, as well as on the soil particles [38]. This reduces the bio-availability and consequently, inhibits the biodegradation to some extent. Even though microbial activity generally slows down at low temperatures, it has been found that many of the crude oils components are degraded at extremely low temperatures. This reflects the adaptation ability of the indigenous soil microorganisms to extreme environments such as in Antarctic and sub-Antarctic climate [39]. The characteristics of a polluted ecosystem can influence the bioremediation process in such a way that, under certain conditions, the crude oil can remain unaltered indefinitely by microorganism, while, under other conditions, the same contaminant can be completely biodegraded within weeks or few months [38].

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Table 2. Biodegradation of oil present in drilling wastes (as % of removal) [33]. Biodegradation (%) Treatments Soil 70 % Drilling waste (10 % oil w/w) Vegetable biomass from tropical trees (20 % ) Fertilizers Soil 70 % Drilling waste (10 % oil w/w) Vegetable biomass from tropical grass (20 % ) Fertilizers Control Soil 70 % Drilling waste (10 % oil w/w) Fertilizers Without Vegetable biomass (bulking agents) 20 28 80 84 80 90 Oil and grease (%) Sat + Arom (%)

Bioremediation experiences in the tropics

Bioremediation approaches have been widely used in the tropics; Venezuela is a typical example where several successful experiences are reported. For instance, a hydrocarbon content reduction from 8 to 0.5% w/w within 130 days has been reported for drilling waste biotreatment [33]. In the Puerto La Cruz region, several pits have been cleaned up with the aid of bioremediation. Specifically, in the Los Nisperitos area we achieved an average 79% w/w crude oil removal (14,246 m3 of oily sludge in 11 pits) after 90 to 120 days. Similarly, 83% w/w hydrocarbon reduction was achieved in the Herradura pit, 80% w/w in the Portn 27 pit (3,044 m3 of oily waste) and in another huge pit where 33,000 m3 were treated [40]. Soils contaminated with a 28 API crude oil spill in the eastern region of Venezuela exhibited a 76% biodegradation rate in 68 days, reaching final total hydrocarbon levels that complied with the Venezuelan environmental regulations [15].

Crude oil properties vs biodegradation rate

As stated above, numerous studies have focused on different variables affecting biodegradation rates of hydrocarbon contaminated soil. Many of them analyze the performance of different microorganisms, whether they are indigenous to the soil or specially cultivated consortia or strains. Others compare the effect of different amendments or nutrients, or the influence of the soil type upon the biodegradation rate. The vast majority of such studies use one specific oil or waste, or even single hydrocarbons or fractions thereof. However, it is the biodegradablilty of the crude oil the single most important parameter that determines the biodegradation rate [41].

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Since each published study uses a particular crude oil and particular set of conditions, it is very difficult to compare results from different studies. Moreover, the contaminating crude oil is very often not well characterized or described, and the definition or assessment of the biodegrdation rate varies from author to author. For example, some studies base their results on direct measurements of the reduction of the whole crude oil content in soil (oil and grease, O&G) while others focus primarily on the saturate and aromatic fractions, neglecting the fate of asphaltenes and resins. Other strategies to assess biodegradation of petroleum hydrocarbons include measurement of CO2 evolution [42] changes in microbial activity, or internal molecular markers [43]. Only few studies have explored the relationship between the crude oils properties and its biodegradability [41, 44-47]. Petroleum is an extremely complex mixture composed mainly of hydrocarbons with the number of carbon atoms ranging from one for trapped methane to far over 150, with no agreement among scientist regarding the upper bound. In addition, it contains molecules with nitrogen, sulfur and oxygen containing functional groups, as well as trace amounts of metals, mainly Ni, V and Fe. Thus, there are millions of different compounds present in crude oil; and this mixture is particular not only for every oil field, but often for every well [48]. The mixture of compounds present in a certain crude oil, and consequently, the oils properties, depend largely upon its history. Original source organic matter, depositional environment, migration patterns, thermal history and exposure to microorganisms in the subsurface are some of the factors that shape the oils composition. Consequently, oils from different geographical regions have different properties; and even oils from a similar region may differ significantly depending on these factors [49]. For instance, the Orinoco Oil Belt, located in the eastern part of the country contains vast reserves of extra heavy petroleum, and not too far, medium petroleum is being produced. Biodegradabilty of a particular oil depends on the susceptibility of each of its components to be degraded by the enzymatic actions of microorganisms. Thus, biodegradability of an oil depends on the oils composition, and varies significantly for different oils. Relative susceptibility to biodegradation among different families of compounds present in crude oils is fairly well established; in many cases, even the reaction pathways are well understood [49]. However, degradation trends change if the compounds are present in complex mixtures or isolated [50] and may vary slightly upon environmental conditions [51]. In general, it is accepted that linear paraffins are readily mineralized by microorganisms [49, 52] followed by branched paraffins, while cycloalkanes (naphthenes) are more recalcitrant; more so, if they are condensed. Hopanes for instance degrade partially only at severe stages of biodegradation and are used as internal indicators to assess the biodegradation extent [43]. Aromatics are more resistant to biodegradation than paraffins; they become more recalcitrant as the number of condensed rings increases. However, polycyclic aromatic molecules with up to 4 condensed rings show significant reduction in their concentration upon biotreatment [53-55]. A recent study even reported that aromatics up to three rings showed better biodegradability than n-alkanes; the same research group reported in another study that PAH (polycyclic aromatic hydrocarbon) were more recalcitrant than alkanes [56, 57]. PAH degradation is of particular importance, since the parent PAHs are regarded as the most toxic substances present in petroleum, and risk based evaluations are based largely on their concentration in the contaminated soil. Alkyl PAHs however, remain long after their non-substituted counterparts have disappeared; among them, different isomers are degraded at different rates [54]. Additionally, biodegradability decreases as molecular weight increases, largely because aromaticity and degree of condensation increase for the larger molecules, even

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though the particular nature and mixture of these large molecules varies significantly among crude oils. Consequently, regardless of their origin, the heavier the crude oil is, the larger the recalcitrant fractions are. This general trend has been established by McMillen et al., who found that there exists a fairly good relationship between API gravity and biodegradability [41] (see Figure 1). The author did not give details regarding the identification of the crude oils used for this model. Surprisingly, despite large potential differences in crude oil properties, as well as in conditions used for biodegradability assays and assessment, we found a fairly
100 90

O&G, Maximum % Loss

80 70 60 50 40 30 20 10 0 0 10 20

y = 2.24x - 19.3 R2 = 0.958

30

40

50

60

API Gravity

Figure 1. Maximum amount of O&G (Oil and Grease) loss during bioremediation for unweathered crude oils in soil and composts versus the API gravity of the crude oil [41].

60
Biodegradation rate (after 90 days)

Biodegradation rate vs API

ME

50 VI 40 30 20 10 0 0 5 AY 10
CA BA y = 2,7655x - 26,669 R2 = 0,9062

SI

GU

BO

TJ 15 API 20 25 30

Figure 2. Maximum amount of O&G (oil and Grease) loss during bioremediation for unweathered Venezuelan crude oils in soil and composts versus the API gravity [47].

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similar trend for nine Venezuelan crude oils, ranging from medium to extra-heavy samples [47] (Figure 2). In this case, microcosms with soils contaminated with 5% w/w of the topped crude oils were submitted to biostimulation over a 120 day time period. There is only a 25% difference between the slopes reported by McMillen, and found in our study. Venezuelan data shows more scattering, and consequently, a poorer correlation coefficient. This might result from the fact that a smaller amount of samples were used, and most of them had API gravities below 20. McMillens results as well as ours seem to indicate that all oils have a recalcitrant fraction that fails to mineralize. Similar results have been observed for hydrocarbon weathering, where the combined action of evaporation, water washing and biodegradation approaches a finite limit [58]. As expected, the recalcitrant fraction is larger for the heavier oils since the larger molecules are the ones that are more resistant towards biodegradation. Data from figures 1 and 2 show that soils contaminated with oils of less than 20 API show little or no net oil and grease reduction upon bioremediation. Several authors have found similar behavior of extra-heavy crude oils towards biodegradation; however, some studies claim significant biodegradation of these types of oils when treated with specially grown inoculums [59-62]. Most of the extra heavy crude oils have already suffered subsurface biodegradation in the reservoir; in fact, this phenomenon is partially responsible for their high density since the smaller and less condensed components have been removed by bacteria, and other compounds have been partially oxidized. Therefore, it is not surprising that they are more recalcitrant towards bioremediation processes than the lighter non subsurface biodegraded oils [52, 58, 63, 64]. Comparison of SARA (Saturates, aromatics, resins and asphaltenes) distribution of the original oil, and the oil recovered from the composts after bioremediation show an increase of the polar fractions, together with a decrease of saturates and aromatics, whereas asphaltene amounts remain largely unchanged. These results indicate that certainly microorganisms partially oxidize saturate and aromatic compounds which now behave as polars but still contribute to the total extracted oil and grease fraction. That is, partial degradation takes place but compounds are not completely mineralized [65, 66]. API gravity is just an average property that exhibits definite trends with other more specific properties like elemental composition, distillation curve, SARA distribution etc. The influence of some of these properties upon biodegradability has been explored in the literature. For crude oils with larger than 13 API, we obtained a better than 0,95 correlation factor for the trend of biodegradability and molar H/C ratio, % of vacuum residue, and T50 (temperature at which 50% of the sample has distilled [47]. Neither sulfur nor trace metal content showed significant trends with biodegradability [47]. Among the SARA families, saturate and aromatic weight content seem to correlate with biodegradability [47]. In Venezuela, SARA analysis has been commonly used to estimate crude oils biodegradability because it is generally accepted that the saturates and aromatics hydrocarbons (including BTEX and the PAHs) are the potentially biodegradable fractions, while the resins and asphaltenes, sometimes grouped as the polar fraction, are much less or not biodegradable at all [67]. We found that indeed an increase in saturates is associated with an increase in biodegradability; however, scattering is significant. Since the saturate fraction includes the readily biodegradable linear alkanes as well as the less biodegradable branched alkanes and the more recalcitrant cycloalkanes, this scattering is actually expected. Opposite to saturates, the biodegradability decreases as the aromatic fraction increases; this trend also shows high scattering. Thus, SARA analysis is not an appropriate tool to predict a crude oils biodegradability.

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In summary, even though API gravity is still the most straightforward indicator to predict biodegradability since this is the most used and simple to measure characterization factor, molar H/C ratio as well as data extracted from the distillation curves may be used to increase accuracy of the prediction. Several authors claim that biodegradability is a matter of bioavailability; therefore, it can be enhanced with the addition of surfactants [68-73]. Even though most publications demonstrate increased biodegradability when using surfactants, none of them achieve a high enough heavy crude oil biodegradability that could be useful for practical applications. A pilot study in Venezuela, where natural surfactants were added to a landfarming area to enhance biodegradation, showed that rather than stimulate the biodegradation, the surfactant promoted the soil washing; that is, O&G lost from the upper layer was found in the underlaying soil [74].

Allowed soil TPH limits: Historical development and scientific support

Before the 1980 decade, the E&P wastes in the US were dewatered and buried to satisfy the land owners demands who received a payment for any damage on the soil [75]. The main impacts of the E&P wastes on the soils were due to the salts, while the impact of the diesel from the drilling mud was considered minor and of short duration [76-77]. In 1986, the State of Louisiana defined the non hazardous character of the E&P wastes (supported by the USEPA in 1988) and established the limit of 1% for the oil & grease content in the soil/waste mixture. For burial, a maximum of 3% oil & grease content was established (Rule 29-b, [78]). The American Petroleum Institute also recommend 1% as the maximum content of TPH in soils from the oilfields [79] and the US States of Texas and Michigan adopted the same limit [80]. A review of the technical evidence utilized to develop the limit of 1% for TPH or O&G content indicates that below this limit no significant plant growth reduction is evident. Any impact derived from oil concentrations between 1 and 5%, disappears after the first vegetative cycle [79]. Recovery of oil impacted sites has been attributed to a combination of abiotic factors (evaporation, sorption on organic matter present in soil, etc) and biodegradation. The impact of the hydrocarbons on soils and plants has been endorsed to physical and biochemical effects [81-83]. An example of a physical effect is the reduction in plant vigor due to the oxygen displacement from the soil pores. Additionally, the sudden availability of carbon causes an explosion of the heterotrophic microorganism growth and depletes the oxygen in the soil. On the other hand, it is well known that the lower molecular weight aromatic compounds that are more water soluble are the more phytotoxic compounds in crude oil and refined products. Therefore, diesel contamination in soil at concentrations as low as 0,1%, may affect crop growth [82, 83]. However, any harmful impact from the diesel is rapidly dissipated after a single vegetative cycle. Recent publications [84] show that the main effect of low TPH concentrations that remain after a natural or an enhanced biodegradation process in the soil is due to physical effects caused by these recalcitrant fractions of the oil. This hydrophobic residue covers the soil particles and severely reduces the soils field capacity. Similarly, porosity, gas exchange and cation exchange properties are altered. The combination of these physical effects results in a net biomass reduction. However, the extent of these effects depends on soils texture and its organic matter content. Sandy soils with low organic C concentration show significant effects even at TPH levels below 1%, while soils with better texture and higher organic C content exhibit a healthy plant development at TPH levels far above 1%. Thus, the authors recommend the establishment of site specific

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residual TPH limits. In addition, Adams and Morales [84] found that recalcitrant TPH fractions present in soil, even at levels above 1%, are not assimilated by plants nor do they pose significant risk for the cattle that grazes in these areas or for people that eat the cattle.

TPH regulation in South America

A quick review of the environmental regulations regarding hydrocarbon contamination in some of the South American oil producing countries shows that the rules are focused on crude oil spills and oily drilling waste treatment from E&P activities. Only minor attention has been devoted to the proper management of hydrocarbon contaminated wastes and spills in downstream activities where the potentially toxic components of the petroleum; that is, BTEX and PAHs, are more concentrated. Venezuela had no practical rules or TPH limits to address the treatment of oil contaminated soils or oily drilling waste until 1996 when a large number of foreign oil companies were invited to explore and develop depleted and unexplored oil fields. Up to that date, the only rule to deal with oily wastes and contaminated soils was the Decree 2211 [86] which established that all wastes from the oil industry are considered hazardous. However there were no legal limits for TPH in treated wastes or soils. Therefore, it was impossible to determine an accepted clean up criteria. Since 1998, Decree 2635 [87] regulates the management of all hazardous wastes; one entire section is devoted exclusively to wastes produced from petroleum E&P and mining activities. The aim of Decree 2635 was to develop a rational approach to manage the E&P wastes and to protect the soil, and resulted from the agreement between the Venezuelan Environmental Ministry, the national oil companies represented by PDVSA (Petrleos de Venezuela) and the representatives of some foreign oil companies. A team of Venezuelan experts was also involved in developing the technical basis of the decree. The general approach for the E&P waste management in Venezuela derives from Louisiana States Rule 29-b [78] According to current regulations, E&P wastes are considered as a special kind of hazardous waste; in practice, this means, that it can be treated and disposed thereafter in the soil. The waste management strategy is aimed to treat and dispose the E&P wastes close to the source (i.e drilling rigs,) to utilize the least possible land area, and to achieve the fastest possible recovery of the soils agrochemical properties. The preferred techniques utilized for these purposes are landspreading for water-based and oil free drilled cuttings, and landfarming and composting for oily drilled cuttings. The last two techniques are bioremediation processes, and in any case, the final destiny of the drilling waste is the soil. Colombia, another South American oil producing country, has no local regulation for the maximum allowed TPH levels in soil. However, Louisianas Rule 29-b is directly applied without any change [88-91]. Thus, Venezuela as well as Colombia consider a 1% O&G content as the end-point for soil treatment to allow spreading, and a 3% O&G to allow burial. Ecuador promulgated the first soil TPH limits in 2001 [92]. This regulation considers three different limits (in ppm of TPH): 1,000, 2,500 and 4,000 for soils or ecosystems considered sensible, of agriculture use and of industrial use respectively, as stated in Table 6 of the Decree 1215 [92]. These limits are not based on any known study or technical evidence. As is the case for Venezuela, the Ecuadorian soil TPH limits do not consider the nature of the hydrocarbons or how weathered they are, nor do they consider the soil properties. Considering that the potentially toxic hydrocarbons are more

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concentrated in fresh and refined products and light crudes, it is evident that this rule is not based on human or environmental risks. In conclusion, the main concern in the South American oil producing countries discussed herein refers to E&P wastes and oilfield spills. Venezuelas and Colombias regulations derive from Louisianas Rule 29-B. The limits established therein obey extensive technical background which basically considers that the hydrocarbons trigger short and long term effects upon the soil. The first ones could include a temporary phytotoxic effect to the vegetation that rapidly disappears due to weathering and biodegradation. No significant long term effect is expected if topsoil TPH is below 1%. Any long term effect, if observed, derives from the hydrophobic properties of the weathered oil and is not related with toxicity. Both countries have adequate rules to deal with E&P oily wastes, but none of them are based on the risks derived from the potentially toxic components of crude oil and refined products (BTEX and PAHs.) Soil TPH limits in Ecuador are more stringent than in Venezuela and Colombia; however there is no known scientific support that justifies those limits.

Soil remediation decision making: When is bioremediation a sound choice?

As was discussed earlier, the extent to which crude oil and oil products biodegrade depends on the biodegradability of their components. The scientific and practical evidence both show that the lighter and more soluble hydrocarbons are rapidly fade in the environment by means of abiotic factors as volatilization, absorption, dilution combined with biodegradation [93-96]. As was mentioned, there is a fairly good relation between the API gravity of the crude oils and its experimental biodegradability measured as the maximum O&G loss (Figure 1). Venezuela produces crude oils that rank from less than 10 API in the Orinoco Oil Belt and Boscn Oil Field to more than 40 API in the Campo Rosario Oil Field. However, most of the production and reserves correspond to heavy and extra heavy crude oils (96); thus, bioremediation does not seem a feasible technique to treat soils impacted with those oils. As an example, Table 3 shows experimental biodegradability test results of nine Venezuelan crude oils that rank from 9 to 28 API [47]. The values are also compared with predicted biodegradability based on API gravity, as suggested by Chevrons model [41]. Considering the differences in experimental procedures to assess biodegradability, the experimental and predicted values match fairly well, with the only exemption is the Sinco crude. Thus, API gravity might yield a reasonable prediction of biodegradability. Table 3 reveals that, regardless of their API gravity, none of the crudes tested could be 100% biodegraded after 120 days of treatment. This is an important fact to consider in Venezuela and Ecuador, since biotreatment is extensively used in both countries for oily waste treatment and oily soil remediation. As stated earlier, popularity of biotreatment arises from its relative simplicity, favorable tropical conditions, the lack of need to use specialized equipment and its low cost as compared to other options. However, often naively high expectations are attributed to biotreatment up to the point that people working in the oil business and environmental authorities in these countries base treatment choices on the false believe that bioremediation can completely vanish O&G or TPH from soil. In the case of the Venezuelan crudes tested (Table 3) the maximum biodegradability found is just 43% for a 28 oAPI crude. However, a significant portion of the oily drilled

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Table 3. Experimental vs predicted O & G loss of selected Venezuelan crudes. Experimental O&G loss (%) NS NS NS NS 19 40 38 42 46 Predicted O&G Loss (%) 1 2 4 8 20 24 37 42 43

Crude Oil Ayacucho Carabobo Bachaquero Ta Juana Boscn Sinco Mesa Guafita La Victoria
a

API 9.0 9.3

10.6 12.3 17.5 19.3 25.0 27.3 28.0

NS: no significant O&G loss was observed. a Crude oil diluted with gasoil

cutting wastes and oily contaminated soils in Venezuela result from E&P activities in the Orinoco Oil Belt area, where the 9 oAPI crudes Ayacucho and Carabobo are produced. These crudes do not show any significant O&G biodegradability; thus, it is obvious that bioremediation of such wastes or soils is not a sound choice. According to the same model, the lightest crude oil from the Ecuadorian oilfields at the Oriente, (30.5 oAPI Lago Agrio crude) [98] would have a maximum biodegradability of about 50%. Table 4 shows the predicted % of residual, non biodegradable O&G or TPH fraction after bioremediation of a hypothetical soil with 2% (20,000 ppm) fresh crude oil for the nine Venezuelan crude oils presented in Table 3, and the Lago Agrio crude from Ecuador. Values in Table 4 show that for the crude oils considered, it is not possible to meet most of the regulatory limits for Oil & Grease or TPH in soils, even at an initial O&G concentration as low as 2%. These predicted residual O&G values suppose that the oil is not weathered. In the latter case, the biodegradability would be even lower than predicted for fresh crude oil. In conclusion, is it possible to accomplish regulatory limits for O&G or TPH in soils using bioremediation? In the authors opinion, the only way is to treat the biodegradable fractions of the oil, and thereafter dilute or disperse the soil that contains the recalcitrant residues to reach the regulatory limit. In the process, the amendments used to enhance biodegradation and to condition the soil or wastes, contribute to the dilution. The lower molecular weight and more soluble compounds that are potentially toxic for humans, animals and plants would indeed be biodegraded, and the recalcitrant fraction would pose no risk. Of course, the remaining TPH residues must be below the level that compromises soil fertility. Some authors claim that the end point for any TPH bioremediation process should be based on specific ecotoxicity tests [99, 100]. However, this approach only makes sense for wastes or contaminated soils with reasonably high biodegradability. Above what oAPI would be reasonable to use biotreatment processes for oily wastes and oil contaminated soils? It depends on soil availability, costs, feasibility of other options, soil use and fertility, local regulations, etc.

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Table 4. Residual O & G after a Bioremediation of a soil with 2% (20,000 ppm) O & G. Meet limits? (Venezuela and Colombia, 10,000 ppm) Meet Ecuadorian limits? Sensitive Ecosystem 1,000 ppm Agriculture use 2,500 ppm Industrial use 4,000 ppm

Crude Oil API Ayacucho Carabobo Bachaquero Ta Juana Boscn Sinco Mesa Guafita La Victoria Lago Agrio 9 9.3 10.6 12.3 17.5 19.3 25 27.3 28 30.5

Predicted O&G loss (%) 1 2 4 8 20 24 37 42 43 50

Residual O&G (ppm) 19,828 19,694 19,111 18,350 16,020 15,214 12,660 11,630 11,316 10,000

No No No No No No No No No Yes

No No No No No No No No No No

No No No No No No No No No No

No No No No No No No No No No

For weathered or heavy crude oil contaminated soils and drilled cuttings there are other more expensive alternatives as their burial or use as a filling material for roads or drilling locations. Other authors claim that phytoremediation could be used as a passive and pulling treatment after a conventional bioremediation process [101].

Phytoremediation
It has been demonstrated that plants could be useful to remediate low to moderately contaminated soil [102]. This fact is related to the ability of roots and their associate microbiota to reduce, contain or render contaminants [103.] Additionally, plant coverage protects soils from erosion, its use is enviromentfriendly and the cost is lower as compared to other physical or chemical treatments. Similarly to other biotreatment techniques, typical warm and almost constant temperatures throughout the year in tropical regions favor plant growing and microflora activity, if water and nutrients are provided in adequate amounts. In Venezuela, research on phytoremediation begun last decade; however, the potential of plants to decontaminate soil has only been tested under greenhouse conditions. Up to the present, most studies have been focused on three aspects: a) plant selection for phytoremediation purposes, b) effects of petroleum contamination on plant germination, survivor, production and root morphology, and c) the ability of selected plants to reduced O&G or TPH in soils. In a preliminary screening to identify plants for phytoremediation purposes, Merkl et al. [104] studied the occurrence of cultivated and indigenous plant species in 4 contaminated

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sites located in Venezuelas eastern lowlands, which are important petroleum producing regions. They found 57 species, comprising 18 legumes, 19 grasses, 3 sedges and 17 others herbaceous species (Table 5) The authors also studied the seed propagation, as well as the tiller and root development of selected species. The aim was to choose the most promising species for soil remediation according their occurrence in different contaminated sites, their abundance, propagation easiness, root system (i.e. fast growing, deep reaching, widely extended roots that create an extended rizosphere) and their life cycle (i.e. perennial rather than annual to avoid the need for yearly reestablishment.) The results show that most of the potentially useful species are indigenous to tropical America, while only one grows worldwide. Out of the 57 identified species, 7 are cultivated and 29 are perennials. Legumes were the group which propagated more easily, whereas most of the grasses and herbaceous species could not be propagated successfully. On the other hand, the most favorable root system belongs to some grasses and sedges, while in general, legumes have less ramified but deeper reaching roots. It has been suggested that commercial grasses could have great potential for phytoremediation due to their fast growth rates and extended and fasciculated root systems which improve the rizospheres ability to degrade contaminants. Additionally, information regarding their establishment, nutrient requirements and maintenance is well known, while seeds are readily available. Mager [105] assessed the seed germination and biomass production of 6 commercial grasses in a silt-sandy soil contaminated with 3% light oil crude. The author found that, after 45 days, the hydrocarbon contamination reduced seed germination and biomass production. Brachiaria brizantha and Panicum maximum attained the highest seed germination rates in contaminated soils, whereas B. brizantha exhibited the highest aboveground and root production and maximum root length (Table 6). Both species were chosen to assess their ability to remediate the contaminated soil and after 240 days. Results plotted in Figure 3 show a final O&G content of 1.0% for soils planted with P. maximum, 1.2% for soils planted with B. brizantha and 1,6% for the control [106]. In another study, Merkl et al. [106] tested the ability of 3 legumes (Calopogonium mucunoides, Centrosema brasilianum and Stylosanthes capitata) and 3 grasses (Brachiaria brizantha, Cyperus aggregatus and Eleusine indica) to phytoremediate a soil contaminated with 5% w/w of heavy crude oil. Under greenhouse conditions, plant biomass production and TPH reduction (total O&G and fractions thereof) were determined after a 90 and a 180 day incubation period. The legumes died before eight weeks and grasses showed a reduced production under the influence of the contaminant. Soils planted with B. brizantha and C. aggregates had a significantly lower final O&G concentration than the control (Figure 4). On the other hand, concentration of saturated hydrocarbons was always lower in planted than unplanted soil, and B. brizantha induced the highest aromatic content reduction, while stimulating microbial population and activity. A positive correlation between root biomass production and oil degradation was found, as well as between oil degradation and root morphology. B. brizantha and C. aggregates showed coarser roots and B. brizantha presented a larger root surface area in contaminated soils (Table 7) [108]. Additionally, a shift of specific root length and surface area per diameter class towards higher diameters was found. Vetiver grass (Vetiveria zizanoides (L.) Nash) has proven to effectively rehabilitate mining affected areas and purify leachate from landfills and eutrophic waters [109, 110 ]. This species adapts to a wide range of edaphic and climatic conditions throughout the tropics and subtropics [109] and has a massive, finely structured, deep-growing root system [111] thus, it is a promising species for petroleum contaminated soil phyto-remediation.

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Table 5. List of herbaceous plant species founded in petroleum contaminated sites of Venezuela (Adapted from Merkl et al. 2004 [104]). Family/Species name Cyperaceae -Cyperus aggregatus (Willd) Endt. -Cyperus compressus L. -Fimbristylis cimosa R. Br. Poaceae -Andropogon fastigiatus Sw. -Anthephora hermaphrodita (L.) Kuntze -Aristida pittieri Henrard -Aristida riparia Trin. -Aristida venezuelae Herard -Axonopus anceps (Mez.) Hitch -Axonopus canescens (Nees) Pilg. -Brachiaria brizantha (Hochst. Ex A. Rich) Stapf.* -Brachiaria decumbens Stapf.* -Brachiaria humidicola (Rendle) Schweick* -Cenchrus pilosus Kunth -Dactyloctenium aegyptium (L.) Willd -Eragrostis ciliaris (L.) R. Bs. -Eragostis guianesis Hitchc. -Eragostis maypurensis (H.B.K.) Stend -Esragostis viscose (Retz.) Trin -Panicum maximum Jacq.* -Pappophorum pappiferum (Lam.) Kuntze -Rhychelytrum repens (Willd) C. E. Hubb Caesalpinaceae -Chamaecrista rotundifolia (Pers.) Greene -Chamaecrista hispiduta (Vahl.) H. S. Irving & Barneby -Chamaecrista flexuosa (L.) Greene Fabaceae -Aeschynomene brasiliana (Poir.) DC -Aeschynomine histrix Poir. -Aeschynomine sp. L. -Desmodium glabrum (Mill.) DC -Cagoloponium mucunoides Desv.* -Centrosema brasilianum (L.) Benth* -Centrosema molle Mart. ex Benth.* -Stylosanthes capitata Vogel* -Stylosanthes guianensis (Aubl.) Sw. -Zornia latifolia Sm. -Zornia filifolia Domin. Life cycle Perennial Annual Annual Annual Annual Perennial Perennial Perennial Perennial Perennial Perennial Perennial Perennial Annual Annual Annual Annual Annual Annual Perennial Perennial Annual Annual Perennial Perennial Perennial Perennial Unknown Perennial Annual Perennial Perennial Perennial Perennial Perennial Perennial World distribution (Sub) Tropical America (Sub) Tropical America, Africa (Sub) Tropical America (Sub) Tropical America, Africa Tropical America Venezuela, Colombia, Guyana From Panama to Brazil Venezuela, Ecuador From Trinidad to Brazil Venezuela (Sub) Tropical America, Africa (Sub) Tropical America, Africa (Sub) Tropical America, Africa Mexico and northern South America (Sub) Tropical America, Africa (Sub) Tropical America Venezuela, Guyana (Sub) Tropical America (Sub) Tropical America, Africa (Sub) Tropical America, Africa Northern South America (Sub) Tropical America, Africa Pantropical (Sub) Tropical America (Sub) Tropical America (Sub) Tropical America (Sub) Tropical America (Sub) Tropical America (Sub) Tropical America Wide distribution (Sub) Tropical America (Sub) Tropical America (Sub) Tropical America (Sub) Tropical America South America No data

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Table 5. Continued Mimosaceae -Mimosa camporum Benth. -Mimosa orthocarpa Spruce -Mimosa somnians Humb &N Bompl. Ex Willd. -Schrankia leptocarpa DC. Compositaceae -Tridax procumbex L. Convolvulaceae -Jacquemontia tamnifolia (L.) Griseb. Euphorbiaceae -Mychostachys corniculata (Vahl) Griseb Lamiaceae -Hyptis sp. Jacq. -Hyptis suaveolens (L.) Poit. Malvaceae -Pavonia cancelata (L.) Cav. -Sida Cordifolia L. -Sida L sp. Passifloraceae -Passiflora foetida L. Rubiaceae -Borreria capitata (R&P) DC -Borreria laevis (Lam.) Griseb -Borreria Sp.G. Mey. -Borreria verticilata (L.) G. Mey. -Diodia teres Walt. Sterculiaceae -Melochia sp. L. -Waltheria indica L. Turneraceae -Pirisqueta viscosa Griseb. *Cultivated species Annual Annual Annual Annual (Sub) Tropical America Mexico, Northern South America (Sub) Tropical America (Sub) Tropical America, Africa

Annual Perennial Annual Unknown Annual Annual Perennial Unknown Perennial Perennial Perennial Unknown Perennial Annual Unknown Perennial Annual

Pantropical (Sub) Tropical America, Africa South America --Pantropical (Sub) Tropical America Pantropical --Pantropical (Sub) Tropical America (Sub) Tropical America Tropical America (Sub) Tropical America --Pantropical Venezuela, Bolivia

However, Brandt et al. [112] tested vetivers performance in a soil contaminated with a heavy crude oil (5%) and found that there were no significant differences in total O&G content of vegetated and unvegetated assays after 6 moths of treatment. Possibly, longer treatment periods are necessary to show enhanced degradation. The results presented above indicate that more research is needed to establish the potential of native and naturalized species, including new species, to remediated petroleum hydrocarbon polluted soils, and use the technique at field scale. Field scale practices require more certainty regarding the plants tolerance to different types and concentrations of petroleum hydrocarbons, as well as their efficiency to remediate contaminated soils under different ecological conditions (i.e. edaphic, climatic, biological constraints). Because phytoremediation efficiency is site-specific and varies significantly

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Table 6. Seed germination, seedling survivor, biomass production and maximum root length after 45 days, for six commercial tropical grasses in soil contaminated with 3% light oil crude [105].
Above ground biomass production (mg/plant) C 82 75 100 100 0 64 NC 81.6 131.2 0.5 107.0 19.2 164.6 C 6.3 5.0 0.4 1.8 0.0 1.8 NC 40.8 67.7 3.8 127.3 6.7 85.5 C 7.5 4.7 1.6 2.7 0.0 1.2 NC 15.9 28.7 0.2 7.3 1.1 32.0 C 3.5 2.6 0.2 1.7 0.0 2.1 Root biomass production (mg/plant) Maximum root length (cm)

Specie

Seed germination (%) NC C 73 27 7 20 13 73

Seedling survivor (%) NC 100 100 50 100 100 93

Brachiaria brizantha Brachiaria decumbens Brachiaria dyctioneura Brachiaria humidicola Cynodon dactylon Panicum maximum

47 60 13 7 13 93

NC: Non-contaminated soil, C: Contaminated soil

Figure 3. Changes in O&G content in soils contaminated with light crude oil (3%) and planted with Panicum maximum and Brachiaria brizantha [106].
6

T O G (% )

Control B. brizantha and C aggregatus B. Brizantha C. aggregatus Control. E. indica E. indica

0 50 100 150 200

Days

Figure 4. Changes in total oil and grease content (O&G) of planted an unplanted soils [106].

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Table 7. Specific root length (SRL), specific root surface area (SRA), average root diameter (ARD) and specific root volume (SRV) standard deviation of graminoids grown in uncontaminated (0%) and heavy crude oil contaminated soil (5%) [108]. Specie B. brizantha C. aggregatus E. indica % 0 5 0 5 0 5 SRL (mg g-1) 244 22 204 25 696 312 410 121 199 70 152 14 SRA (m2 kg-1) 969 84 1313 167* 1668 759 1408 406 108 24 85 10 ARD (mm) 1.30 0.07 2.09 0.46* 0.84 0.07 1.19 0.14* 0.19 0.02 0.l9 0.03 SRV (dm3 g-1) 3455 460 7684 2557 3893 1895 4296 1105 52 4 43 9

*Values differ significantly from corresponding control (p< 0.05, Students t test).

with environmental factors, issues like nutrient supply and adequate C:N:P ratios, soil textures and humidity, and diverse bulking agents should be considered in future studies. For proper assessment of phytoremediation efficiency, it is important to establish the mass balance in order to quantify how much TPH is volatilized, retained in the soil or absorbed by plants. Presently, several studies are being carried out with different contaminants like crude oil, refined products (i.e. diesel, gasoline), and oil impregnated drilled cuttings. Additionally, phytoremediation has been assayed as a sole clean-up technique, or in combination with landfarming. In the latter case, initial decontamination is accomplished though landfarming, followed phytoremediation (phased bioremediation) to treat the more recalcitrant fractions. As stated above, these fractions are little or not toxic; thus, plants can grow more vigorously and develop and extensive rhizosphere needed for effective decontamination and soil stabilization. In conclusion, although biological treatments are effective for the remediation of soils contaminated with medium and light crude oils, much development is still needed to treat heavy and extra-heavy oil contamination, considering that these are the more abundant petroleum types, and their production will increase as long as fossil energy remains the leading energy source.

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