Appendix 4

SAMPLE LITERATURE

REVIEW

The following literature review was written in conjunction with the proposal in Appendix 5. As any good literature review, it serves (1) as an introduction to the topic, (2) as background information to make the topic more meaningful to the researcher as well as the reader, and (3) as justification for a study. Similar to the proposed study itself, it introduces the problem (polycyclic aromatic hydrocar- bon contamination) and possible solutions to the problem (dissipation, remedi- ation), with emphasis on the solution proposed in the study (phytoremediation with microorganisms in the rhizosphere). Organization and content for the review are focused around these key issues, which can be used as the key words for the literature search that provided the content. Note the review is not just an annotated listing of reports published in the area but a smooth-flowing discus- sion of the issues with appropriate references interspersed. See how Mr. Gentry moved from the general subject to those areas specifically related to his own and ends with a concluding justification for his own study. Also note how he has used headings and transitions to lead the reader through the review. (This review of literature, the proposal in Appendix 5, and an accompanying slide set depicted in Appendix 13 are the work of Terry J. Gentry, a master's candidate at the time he produced this work, now Dr. Terry J. Gentry with Oak Ridge National Laboratory in Oak Ridge, TN. All are used with his permission.)

P O L Y C Y C L I C A R O M A T I C H Y D R O C A R B O N I N F L U E N C E O N

R H I Z O S P H E R E M I C R O B I A L E C O L O G Y

Contamination of soil by toxic organic chemicals is widespread and frequent. This is sometimes the result of large-scale incidents such as the Exxon Valdez

261

262 Appendix 4

oil spill in Alaska (Pritchard and Costa, 1991). But, more often, smaller areas of soil are polluted. Cole (1992) estimated that in the United States there are 0.5 to 1.5 million underground storage tanks leaking into the surrounding soft. In situ

bioremediation of these contaminated sites may be more feasible than chemical and physical clean-up methods. Degradation of polycyclic aromatic hydrocarbons (PAHs), a major constituent of many of these pollutants, can be possible if PAH- degrading microorganisms are present at the site. These microorganisms may be more prolific in the rhizosphere of plants than in soil with no vegetation.

A. Polycyclic Aromatic Hydrocarbons (PAHs)

Polycyclic aromatic hydrocarbons are organic compounds that are typically toxic and recalcitrant (Sims and Overcash, 1983). They consist of at least three benzene tings joined in a linear, angular, or cluster array (Cerniglia, 1992). Edwards (1983) described PAHs as being practically insoluble in water. They are produced by various processes, including the incomplete combustion of organic compounds such as petroleum (Giger and Blumer, 1974; Laflamme and Hites, 1978). The carcinogenicity of many PAHs has been well documented (Haddow, 1974). This knowledge has prompted much research to determine the mode by which these compounds cause cancer and their ultimate health risks to humans (Miller and Miller, 1981). Due to their toxic nature, the United States Environmental Protection Agency included several PAHs in their list of priority pollutants to be monitored in industrial wastewaters (Keith and Telliard, 1979). Heitkamp and Cerniglia (1988) concluded that this interest has resulted in increased efforts to remediate PAH-contaminated soil.

B. Dissipation

Reilley et al. (1996) reported the fate of PAHs in soil includes irreversible sorption, leaching, accumulation by plants, and biodegradation. They also con- tended that surface adsorption is the main process controlling PAH destination in soil. Many PAHs are strongly adsorbed to soil particles (Knox et al., 1993). Means et al. (1980) found the PAHs composed of longer chains and greater masses to be more strongly adsorbed to soil particulate matter. Leaching of PAHs from soil is minimal due their adsorption to soil particles and low water solubility (Reilley et al., 1996). Results indicate that larger PAHs may adsorb onto roots, but translocation from roots to foliar portions of the plants is negli- gible (Edwards, 1983; Sims and Overcash, 1983). Biodegradation is the main pathway by which dissipation can be enhanced.

C. Bioremediation

Bioremediation manipulates biodegradation processes by using living organisms to reduce or eliminate hazards resulting from accumulation of toxic chemicals

Sample Literature Review .263

and other hazardous wastes. According to Bollag and Bollag (1995), two tech- niques that may be used in bioremediation are (1) stimulation of the activity of the indigenous microorganisms by the addition of nutrients, regulation of redox conditions, optimization of pH, or augmentation of other conditions to produce an environment more conducive to microbial growth and (2) inoculation of the contaminated sites with microorganisms of specific biotransforming abilities.

1. Indigenous Population. Soil contains a large and diverse population of microorganisms (Tiedje, 1994). The indigenous population of these microor- ganisms has been manipulated to increase biodegradation. In situ bioremedia- tion utilizes organisms at the site of pollution to remove contaminants. Often, indigenous organisms from the contaminated area, which may even have adapted to proliferate on the chemical, are utilized to remove the pollutants (Bollag and Bollag, 1995).

Microbial degradation may be enhanced by aeration, irrigation, and application of fertilizers (Lehtom~_ki and Niemel~i, 1975). In Prince William Sound, Alaska, following the Exxon Valdez oil spill, the application of fertilizers increased biodegradation up to threefold (Pritchard and Costa, 1991).

The relative contributions of bacterial and fungal populations to hydrocarbon mineralization may differ based upon contaminant and soil parameters. Anderson and Domsch (1975) studied the degradation of glucose in several soils. They attributed the majority of mineralization to fungi (60%-90%) with relatively minor bacterial contribution (10%-40%). It is unclear if fungi are also the prin- cipal degraders of hydrocarbons (Bossert and Bartha, 1984). Song et al. (1986) reported 82% of n-hexadecane mineralization was due to bacteria while fungi contributed only 13%. They concluded that bacteria are the primary degraders of n-hexadecane in the soil tested, but additional experiments are necessary before the results can be generalized. In a field study utilizing six oils as con- taminants, Raymond et al. (1976) noted that fungi appeared to be the principal hydrocarbon-degraders.

From a review of the literature, Cerniglia (1992) found various bacteria, fungi, and algae reported to degrade PAHs. More specifically, Dgziel et al. (1996) isolated 23 bacteria capable of utilizing naphthalene and phenanthrene as their sole growth substrate. These bacteria were all fluorescent pseudo- monads. Shabad and Cohan (1972) reported that soil bacteria are the primary degraders of PAHs. Cerniglia's (1992) review concluded that the microbial degradation of smaller PAHs such as phenanthrene has been thoroughly inves- tigated; however, there has not been sufficient research on the microorganisms capable of degrading PAHs containing four or more aromatic tings. There remains a need for isolation and identification of microorganisms capable of degrading the more persistent and toxic PAHs (Cerniglia, 1992).

2. Introduced Microorganisms. Organisms capable of breaking down certain pollutants are not present at all sites; therefore, inoculation of the soil with

264 Appendix 4

microorganisms, or bioaugmentation, is sometimes required (Alexander, 1994). Indigenous or exogenous microorganisms may be applied to the polluted soil (Turco and Sadowsky, 1995). Microorganisms capable of degrading several pol- lutants including PAHs have been isolated from contaminated soil (Heitkamp and Cerniglia, 1988). In addition, Lindow et al. (1989) communicated a need for the continued development of genetically engineered microorganisms includ- ing those capable of degrading a variety of pollutants. Nevertheless, successful establishment of introduced microorganisms remains enigmatic (Pritchard, 1992; Turco and Sadowsky, 1995). Thies et al. (1991) linked the poor survival of intro- duced microorganisms to competition from native soil microorganisms.

The characteristics that allow introduced microorganisms to become accli- mated to a new environment have not been completely elucidated (Turco and Sadowsky, 1995). However, the indigenous soil populations appear to have specific qualities, such as the ability to utilize a particular growth substrate, that give them a competitive advantage in occupying available niches (Atlas and Bartha, 1993). One way to encourage the growth of introduced microorganisms may be to supply a new niche for microbial growth in the form of a suitable plant.

D. Phytoremediation

Phytoremediation is defined by Cunningham and Lee (1995) as "the use of green plants to remove, contain, or render harmless environmental contami- nants." This applies to all plant-influenced biological, microbial, chemical, and physical processes that contribute to the remediation of contaminated sites (Cunningham and Berti, 1993). Plants have historically been developed for food or fiber production. With an increasing interest in the use of plants to reduce contamination from organic chemicals, plants may be selected and developed based upon their suitability for bioremediation. Cunningham and Lee (1995) contend that plant attributes such as rooting depth, structure and density can be altered to increase biodegradation. They assert that, if contaminants are (1) in the upper portion of the soil, (2) resistant to leaching, and (3) not an immediate hazard, many may be removed by phytoremediation. Experiments may confirm that phytoremediation is a less expensive, more permanent, and less invasive technique than many current methods of remediation (Cunningham and Lee, 1995).

E. The Rhizosphere

Curl and Truelove (1986) have described the rhizosphere as the zone of soil under the direct influence of plant roots and in which there is an increased level of microbial numbers and activity. They report that the ratio of bacteria and

Sample Literature Review 265

fungi in the rhizosphere to the non-rhizosphere soil (R/S ratio) commonly ranges from 2 to 20 due to the root exudation of easily metabolizable substrates. These exudates include sugars, amino compounds, organic acids, fatty acids, growth factors, and nucleotides (Curl and Truelove, 1986). Legumes usually demonstrate a greater rhizosphere effect than non-legumes (Atlas and Bartha, 1993). Also, the development of plant roots in previously nonvegetated soil may alter soil environmental conditions including carbon dioxide and oxygen con- centrations, osmotic and redox potentials, pH, and moisture content (Anderson and Coats, 1995).

Generally, the rhizosphere is colonized by a predominantly gram-negative bacterial community (Curl and Truelove, 1986). Anderson and Coats (1995) reported that one of the interesting and repeated topics discussed during the 1993 American Chemical Society symposium was the prevalence of gram-negative microorganisms in the rhizosphere. Reportedly, the ability of gram-negative bacteria to quickly metabolize root exudates contributes to their predominance in the rhizosphere (Atlas and Bartha, 1993).

Anderson and Coats (1995) suggest that increased rates of contaminant degradation in the rhizosphere compared to nonvegetated soil may result from increased numbers and diversity of microorganisms.

1. Rhizosphere Effect on PAHs. The rhizosphere of numerous plants has been reported to increase the biodegradation of several PAHs. Aprill and Sims (1990) examined the effects of eight prairie grasses (Andropogon gerardi, Sorghastrum nutans, Panicum virgatum, Elymus canadensis, Schizachyrium scoparius, Bouteloua curtipendula, Agropyron smithii, and Bouteloua gracilis) on the biodegradation of four PAHs, benzo(a)pyrene, benz(a)anthracene, chrysene, and dibenz(a,h)anthracene. They reported significantly greater dis- appearance of the PAHs in the vegetated soils compared to the unvegetated soils, and the rate of disappearance was directly related to the water solubility of each compound.

Reilley et al. (1996) investigated the rhizosphere effect of alfalfa (Medicago sativa L. ), fescue (Festuca arundinacea Schreb.), sudangrass (Sorghum vulgate L. ), and switchgrass (Panicum virgatum L.) on the degradation of pyrene and anthracene. They reported that the vegetation significantly increased the degra- dation of these PAHs in the soil. They concluded that degradation most likely resulted from an elevated microbial population in the rhizosphere due to the presence of root exudates.

Nichols et al. (1996) conducted an experiment on the degradation of a model organic contaminant (MOC) composed of six organic chemicals including two PAHs (phenanthrene and pyrene) in the rhizospheres of alfalfa (Medicago sativa, var. Vernal) and alpine bluegrass (Poa alpina). They found increased numbers of hydrocarbon-degrading microorganisms in the rhizospheres of both plants.

266 Appendix 4

From the same study, Rogers et al. (1996) reported that plants demonstrated no significant impact on the degradation of the M OC. They concluded it was probable that biological and/or abiotic processes occurring before plants devel- oped enough to produce a rhizosphere effect were responsible for the disap- pearance of the M OC compounds.

2. Rhizosphere microbial ecology in PAH-contaminated soil. Before microorganisms can be successfully introduced into the soil or managed for increased bioremediation, an increased understanding of the determinants of rhizosphere microbial ecology needs to be developed. Anderson and Coats (1995) stated the need for an expanded understanding of the interactions between plants, microorganisms, and chemicals in the root zone in order to identify conditions where phytoremediation using rhizosphere microorganisms is most feasible.

To date, no studies have been conducted on rhizosphere microbial ecology in PAH-contaminated soil. Furthermore, little is known about the factors con- trolling rhizosphere microbial ecology in uncontaminated soil. Bowen (1980) asserted the plant to be the predominant force in the rhizosphere system. In contrast, Bachmann and Kinzel (1992) reported that, in a study involving six plants and four soils, the soil was the dominant factor in some plant-soil combinations. A unique symbiosis that developed from the combination of a specific plant and soil microorganisms was evident. Of all tested plants, Medicago sativa had the strongest influence on the soil. They concluded that this effect was consistent with the results of Angers and Mehuys (1990) and may be related to the nitrogen- fixing activity of alfalfa.

Additionally, recent research suggests that gram-positive bacteria may be a larger component of the rhizosphere microbial population than previously reported. Cattelan et al. (1995) found a large percentage of soybean (Glycine max) rhizosphere population to be occupied by the gram-positive bacterial genus Bacillus spp. Also, it appears that gram-positive microorganisms may play a major role in the breakdown of contaminants including PAHs. Heitkamp and Cerniglia (1988) isolated a gram-positive bacterium capable of degrading several PAHs. The bacterium could not utilize PAHs as the sole C source, but it did completely mineralize PAHs when supplied with common organic carbon sources such as peptone and starch. Additional research is needed to eluci- date the determinants of rhizosphere microbial ecology especially in PAH- contaminated soils.

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Angers, D. A., and Mehuys, G. R. (1990). Barley and alfalfa cropping effects on carbohydrate contents of a clay soil and its size fractions. Soil Biol. Biochem. 22, 285-288.

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