002- Hydrocarbon Bioremediation and Phytorremediation in Tropical Soils

7/30/2019 002- Hydrocarbon Bioremediation and Phytorremediation in Tropical Soils

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Research Signpost37/661 (2), Fort P.O.

Trivandrum-695 023Kerala, India

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

25. Hydrocarbon bioremediation and

phytoremediation in tropical soils: Venezuelan

study case

Infante, C.1, Morales, F.2, Ehrmann, E. U.2, Hernndez-Valencia, I.3 and Leon, N.41Instituto 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;

4

COPRESA. Consultora Ambiental

Abstract. Bioremediation has been widely used in tropical oil producingcountries to remediate oil contaminated soils, crude oil pit bottoms and to treatoily wastes like drilled cuttings and oily tank bottoms. This paper presents a

brief review of some of the most relevant research and field bioremediationstudies in Venezuela. In agreement with international publications, lab results aswell as field experience show that for open bioremediation processes, likelandfarming and composting, biostimulation of indigenous microbial population

is always more effective than bioaugmentation. Moreover, the success of anyopen bioremediation process depends largely on the physical and chemicalconditioning 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/phosphorusratios, among other key factors. Similarly to results from other countries,experimental findings in Venezuela show that crude oil and oil products are not100% biodegradable. Oil properties like API gravity, the content of aromatic

and polar fractions, H/C ratio, distillation residue, among others, determine oilbiodegradability. For instance, low API gravity crude oils show negligiblebiodegradability. Consequently, bioremediation is not a sound choice to treat

heavy crude oil contaminated soils or heavy crude oil drilled cuttings. Bioremediationremoves the more toxic and lower molecular weight hydrocarbons. The remainingrecalcitrant TPH (Total petroleum hydrocarbon) fractions are much less or nottoxic 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: [email protected]


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Infante, C. et al.430

of local cleanup criteria and/or the allowable TPH levels in soils after completion of thebioremediation 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 alsopresents 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 thatcould 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 insoils, 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 significantdiscoveries 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, spillcontaminated soils and tank bottom sludge. Historically, these wastes used to beconfined 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 bioremediationthroughout the year. Not surprisingly, to date bioremediation is the most widely used

technique to clean up oil contaminated soils in Venezuela. Nevertheless, its application islimited 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 microbialactivity 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 tropicalregions.

Biodegradation and bioremediation

Biodegradation is the partial simplification (transformation) or complete destruction

(mineralization) of the molecular structure of environmental pollutants throughphysiological 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 contaminantsto 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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Hydrocarbon bioremediation and phytoremediation in Venezuela 431

specifically applies to the remediation of contaminated soil. The term was coined in the80s, 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 biologicalpreparations (inoculums,) grown in a laboratory and added in large quantities to the field.The microorganism activity is enhanced through addition of suitable bulking agents aswell 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 beeither specific bacteria or fungi strains, or mixtures thereof. Both, fungi and bacteria, playimportant roles in hydrocarbon biodegradation [6] none of them can be regarded as morerelevant than the other. Nevertheless, there are significant differences between the fungiand bacteria hydrocarbon metabolisms [7, 8]. Relative dominance of one species over theother 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. Forinstance, it has been reported that in the tropics a higher diversity of microorganisms ableto readily degrade hydrocarbon compounds is ubiquitously present in the soil. Amongother reasons, the steady and warm temperatures throughout the year in tropical regionsmay 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 areseveral reasons why bioaugmentation is not the best choice. First of all, it is difficult tocultivate a representative population of bacteria and fungi capable of degradinghydrocarbons because it has been found that just over 1% of total microorganism

population present in soil can grow under laboratory conditions. It is virtually impossibleto reproduce the physiology and metabolic interactions among populations of bacterialspecies 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 viewand are regulated by the microclimatic conditions of each soil, which cannot be recreatedin the laboratory. Even though molecular biology techniques have allowed majoradvances 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 mightnot grow under laboratory conditions; thus, the interactions present among thecommunities in the original substrate are precluded in the laboratory culture [12]. Theseinteractions may be relevant for the degradation process and their absence maycompromise the success of the remediation. Additionally, the variety of compounds

present in petroleum, comprising molecules with aromatic, aliphatic and heteroatomicmoieties can only be degraded by the concerted action of different microbialcommunities. As a result of all the above mentioned factors, commercially availablemicrobial 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 predationand competition with the indigenous microorganisms, the introduced cultures willdisappear faster; therefore, continuous addition of the foreign consortia is mandatory forsuccessful clean-up [13, 14].

Experience in Venezuela has shown that biostimulation is a better option than

bioaugmentation. For instance, table 1 compares bioaugmentation vs bioestimulationresults 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 assayresults. 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 m2plots with 10% w/w crude/soil ratios of the same oily sludge used for thelaboratory 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 helpof 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


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excessively large carbon source; thus, relative nitrogen and phosphorus availability is tooshort 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/Pratio 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 inimproving 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 pooroxygen 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 claysoil 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 porosityof 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 organiccompounds increase. This phenomenon results in a larger pollutant water interface which

enhances the pollutant microorganism contact and thus, the oil degrading microorganismcolonization. However, the same physical phenomenon can lead to greater hydrocarbonabsorption 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 toextreme 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 unalteredindefinitely 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].

Treatments

Biodegradation (%)

Oil and grease (%) Sat + Arom (%)

Soil 70 %

Drilling waste (10 % oil w/w)

Vegetable biomass from tropical trees (20 % )

Fertilizers

80 90

Soil 70 %


Vegetable biomass from tropical grass (20 % )

Fertilizers

80 84

Control

Soil 70 %


Fertilizers

Without Vegetable biomass (bulking agents)

20 28

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 fordrilling 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 spillin the eastern region of Venezuela exhibited a 76% biodegradation rate in 68 days,

reaching final total hydrocarbon levels that complied with the Venezuelan environmentalregulations [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 orspecially 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 singlehydrocarbons 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 assessbiodegradation 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 propertiesand its biodegradability [41, 44-47]. Petroleum is an extremely complex mixture

composed mainly of hydrocarbons with the number of carbon atoms ranging from one fortrapped 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 notonly 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, oilsfrom 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 extraheavy 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 uponenvironmental 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. Hopanesfor instance degrade partially only at severe stages of biodegradation and are used as

internal indicators to assess the biodegradation extent [43]. Aromatics are more resistantto biodegradation than paraffins; they become more recalcitrant as the number of condensedrings 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, andrisk 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 significantlyamong 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, aswell as in conditions used for biodegradability assays and assessment, we found a fairly

y = 2.24x - 19.3

R2

= 0.958

0

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0 10 20 30 40 50 60

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O&G,Maximum%Loss

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= 0.958

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

Biodegradation rate vs API

y = 2,7655x - 26,669R2 = 0,9062

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Biodegradation rate vs API

y = 2,7655x - 26,669R2 = 0,9062

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Figure 2. Maximum amount of O&G (oil and Grease) loss during bioremediation for unweatheredVenezuelan 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-heavysamples [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 andbiodegradation 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 resistanttowards 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 ofoils 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 towardsbioremediation 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 anincrease 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 nowbehave 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 inthe 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]. Neithersulfur nor trace metal content showed significant trends with biodegradability [47].

Among the SARA families, saturate and aromatic weight content seem to correlatewith biodegradability [47]. In Venezuela, SARA analysis has been commonly used toestimate 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 biodegradablelinear 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 showshigh 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 topredict 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 ahigh 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 thebiodegradation, 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 tosatisfy 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 theimpact 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 theUS 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. Anyimpact 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 reductionin 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 lowermolecular weight aromatic compounds that are more water soluble are the more phytotoxic

compounds in crude oil and refined products. Therefore, diesel contamination in soil atconcentrations as low as 0,1%, may affect crop growth [82, 83]. However, any harmfulimpact 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 physicaleffects 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 withbetter 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 TPHfractions 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 hydrocarboncontamination 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 hydrocarboncontaminated 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 Decree2211 [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 miningactivities. 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 fromLouisiana 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 thefastest 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 arebioremediation 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 ecosystemsconsidered 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 notconsider 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


12/22


concentrated in fresh and refined products and light crudes, it is evident that this rule isnot 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 temporaryphytotoxic 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 theweathered 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 thepotentially 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 themaximum 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 resultsof 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 inVenezuela 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 usespecialized 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 biodegradabilityfound is just 43% for a 28 oAPI crude. However, a significant portion of the oily drilled


13/22


Table 3. Experimental vs predicted O & G loss of selected Venezuelan crudes.

Crude Oil oAPIExperimental O&G

loss (%)Predicted O&G Loss

(%)

Ayacucho 9.0 NS 1

Carabobo 9.3 NS 2

Bachaquero 10.6 NS 4

Ta Juana 12.3 NS 8

Boscna 17.5 19 20

Sinco 19.3 40 24

Mesa 25.0 38 37

Guafita 27.3 42 42

La Victoria 28.0 46 43

NS: no significant O&G loss was observed.aCrude 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 thatbioremediation 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 meetmost 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 predictedfor 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 biodegradablefractions of the oil, and thereafter dilute or disperse the soil that contains the recalcitrantresidues 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 poseno 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.


14/22


Table 4. Residual O & G after a Bioremediation of a soil with 2% (20,000 ppm) O & G.

Crude Oil

API

PredictedO&G

loss (%)

ResidualO&G

(ppm)

Meetlimits?

(Venezuelaand

Colombia,10,000ppm)

Meet Ecuadorian limits?

SensitiveEco-

system1,000ppm

Agricul-

ture use

2,500

ppm

Indus-

trial use

4,000

ppm

Ayacucho 9 1 19,828 No No No No

Carabobo 9.3 2 19,694 No No No No

Bachaquero 10.6 4 19,111 No No No No

Ta Juana 12.3 8 18,350 No No No No

Boscn 17.5 20 16,020 No No No No

Sinco 19.3 24 15,214 No No No No

Mesa 25 37 12,660 No No No No

Guafita 27.3 42 11,630 No No No No

La Victoria 28 43 11,316 No No No No

Lago Agrio 30.5 50 10,000 Yes No No No

For weathered or heavy crude oil contaminated soils and drilled cuttings there areother 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 tomoderately 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 enviroment-friendly and the cost is lower as compared to other physical or chemical treatments.

Similarly to other biotreatment techniques, typical warm and almost constanttemperatures 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 potentialof 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 plantgermination, 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


15/22


sites located in Venezuelas eastern lowlands, which are important petroleum producingregions. 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 lifecycle (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 arecultivated and 29 are perennials. Legumes were the group which propagated more easily,

whereas most of the grasses and herbaceous species could not be propagatedsuccessfully. 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 rootsystems 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 contaminationreduced 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 withB. brizantha and 1,6% for thecontrol [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, plantbiomass 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 saturatedhydrocarbons was always lower in planted than unplanted soil, andB. brizantha inducedthe 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 andB. 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 thetropics 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.


16/22


17/22


Table 5. Continued

Mimosaceae

-Mimosa camporum Benth.

-Mimosa orthocarpa Spruce

-Mimosa somnians Humb &N Bompl. ExWilld.

-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.

Annual

Annual

Annual

Annual

Annual

Perennial

Annual

Unknown

Annual

Annual

Perennial

Unknown

Perennial

Perennial

Perennial

Unknown

Perennial

Annual

Unknown

Perennial

Annual

(Sub) Tropical America

Mexico, Northern South America


(Sub) Tropical America, Africa

Pantropical

(Sub) Tropical America, Africa

South America

---

Pantropical


Pantropical

---

Pantropical



Tropical America


---

Pantropical

Venezuela, Bolivia

*Cultivated species

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, longertreatment 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 scalepractices 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, biologicalconstraints). Because phytoremediation efficiency is site-specific and varies significantly


18/22


Table 6. Seed germination, seedling survivor, biomass production and maximum root length after45 days, for six commercial tropical grasses in soil contaminated with 3% light oil crude [105].

Specie

Seed

germination

(%)

Seedling

survivor

(%)

Above ground

biomass

production

(mg/plant)

Root biomass

production

(mg/plant)

Maximum

root length

(cm)

NC C NC C NC C NC C NC C

Brachiaria brizantha

Brachiaria decumbens

Brachiaria dyctioneura

Brachiaria humidicola

Cynodon dactylon

Panicum maximum

47

60

13

7

13

93

73

27

7

20

13

73

100

100

50

100

100

93

82

75

100

100

0

64

81.6

131.2

0.5

107.0

19.2

164.6

6.3

5.0

0.4

1.8

0.0

1.8

40.8

67.7

3.8

127.3

6.7

85.5

7.5

4.7

1.6

2.7

0.0

1.2

15.9

28.7

0.2

7.3

1.1

32.0

3.5

2.6

0.2

1.7

0.0

2.1

NC: Non-contaminated soil, C: Contaminated soil

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

0

1

2

3

4

5

6

0 50 100 150 200

Days

TOG

(%

Control B. brizantha and C aggregatus

B. Brizantha

C. aggregatus

Control. E. indica

E. indica

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


19/22


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 % SRL (mg g-1) SRA (m2 kg-1) ARD (mm) SRV (dm3 g-1)

B. brizantha

C. aggregatus

E. indica

0

5

0

5

0

5

244 22

204 25

696 312

410 121

199 70

152 14

969 84

1313 167*

1668 759

1408 406

108 24

85 10

1.30 0.07

2.09 0.46*

0.84 0.07

1.19 0.14*

0.19 0.02

0.l9 0.03

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 orabsorbed 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-uptechnique, 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 moreabundant petroleum types, and their production will increase as long as fossil energy

remains the leading energy source.

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