Introduction

Treatability assessments are used to identify limitations to the rate or endpoint of bioremediation for a specific soil-contaminant combination. For treatability studies, the degradation pathways for the contaminant are generally known (see Chapter 4, Section 4.2.1), but the limitations in a particular soil or at a specific site are less well understood. The tremendous utility of treatability studies is in evaluating practical treatment regimes prior to full-scale implementation. The goal is to demonstrate practicability, optimize treatment design, and provide information for project planning. Sometimes this is an essential proving step for clients or regulators because choice of treatment depends primarily on urgency of remediation and cost. The cost-time relationship for different treatment types is illustrated in Chapter 1, Figure 1.1. The ability to predict the rate of bioremediation progress for a treatment scheme is particularly important in cold regions where costs are higher and treatment times are longer than in temperate regions.

In an effort to understand and improve the bioremediation process in cold regions, researchers have used treatability experiments to:

• identify the presence or absence of microbial activity for a particular contaminant or group of contaminants;

• determine optimum requirements, such as temperature, nutrients, oxygen, and water, for bacteria and fungi to metabolize contaminants in the soil regime;

• examine the effects that natural cycles, such as freezing-thawing and wetting-drying, have on microbial activity and degradation rate;

• estimate achievable endpoints;

• predict and compare treatment times and costs.

Treatability studies can involve in vitro microcosms with individual bacterial species or consortia from the soil incubated in liquid or slurry media, mesocosm studies with soils and natural microfauna, or field trials.

Introduction

Oil and fuel spills are among the most extensive and environmentally damaging pollution problems in cold regions and are recognized as potential threats to human and ecosystem health. It is generally thought that spills are more damaging in cold regions, and that ecosystem recovery is slower than in warmer climates (AMAP 1998; Det Norske Veritas 2003). Slow natural attenuation rates mean that petroleum concentrations remain high for many years, and site managers are therefore often forced to select among a range of more active remediation options, each of which involves a trade-off between cost and treatment time (Figure 11). The acceptable treatment timeline is usually dictated by financial circumstance, perceived risks, regulatory pressure, or transfer of land ownership.

In situations where remediation and site closure are not urgent, natural attenuation is often considered an option. However, for many cold region sites, contaminants rapidly migrate off-site (Gore et al. 1999; Snape et al. 2006a). In seasonally frozen ground, especially in wetlands, a pulse of contamination is often released with each summer thaw (AMAP 1998; Snape et al. 2002). In these circumstances natural attenuation is likely not a satisfactory option. Simply excavating contaminants and removing them for off-site treatment may not be viable either, because the costs are often prohibitive and the environmental consequences of bulk extraction can equal or exceed the damage caused by the initial spill (Filler et al. 2006; Riser-Roberts 1998).

Introduction

Movement of petroleum through non-freezing soils has been studied extensively over the last several decades. Little work has been done on understanding how petroleum moves through seasonal freezing soils (active layer) and frozen soil (permafrost). Petroleum migration through active layer and permafrost soils is influenced by the formation and presence of ice at all scales. At the millimeter scale, ice in pore spaces will either interrupt downward migration causing petroleum to spread laterally, or impede petroleum movement altogether due to the lack of open pore space. Segregated ice at centimeter-to-meter scales will most likely cause the contamination to spread laterally in frozen soils. Segregated ice formation in the active layer can also generate fissures that will enhance petroleum movement when the soil is thawed. At larger scales, discontinuous and continuous permafrost will slow, redirect, or impede contaminant migration.

Understanding the impact freezing and frozen soil conditions have on petroleum movement through soils is necessary to regulation, assessment, and cleanup of contaminated soil and groundwater. A good example of this impact is provided when considering natural attenuation. Seasonal ice and post-cryogenic structure present in active layer soil will influence the movement of petroleum and dissolved compounds, thereby impacting the design of monitoring systems to track natural attenuation. Moreover, cold soil temperatures will slow the physical weathering of compounds in the subsurface. Cleanup levels established for cold regions contaminated soil (Chapter 1) and any remediation plan developed for these sites must account for these impacts.

Bioremediation of Petroleum Hydrocarbons in Cold Regions is written by a multi-disciplinary group of scientists and engineering professionals working in polar regions. The monograph is designed as a state-of-the-art guidance book to assist industry, environmental practitioners, and regulators with environmental cleanup in cold climates. The book can also be used for environmental science and remediation engineering seniors and graduate students who are preparing for a career in professional environmental practice or applied scientific research. The intent of this book is to articulate conditions unique to our cold regions, and present practical and cost-effective remediation methods for removing petroleum contamination from tundra, taiga, alpine, and polar terrain.

Oil and its refined products represent a significant proportion of the pollution found in the Arctic and Antarctic. This pollution is encountered at former military and industrial sites, scientific research stations, rural communities, and remote airstrips, while recent spills and releases tend to be associated with resource development and transportation mishaps. Bioremediation is recognized as potentially the most cost-effective technology for removing petroleum contaminants from ecosystems in cold regions.

Permafrost, suprapermafrost water, tundra, cold-tolerant microorganisms, short summers and long, dark winters, cold air and ground temperatures, and annual freezing and thawing of the active layer are but a few environmental characteristics of cold regions. Their prevalence limits practical remediation methods and has led to the development of innovative and pragmatic bioremediation schemes for use at contaminated sites in cold climates.

Introduction

In order to demonstrate the effectiveness of a bioremediation project, one must have an accurate measure of the contaminants, both at the start of the project and throughout the treatment process. The measurement of the contaminants throughout the process is important to demonstrate that the treatment is successful and to identify advances or set-backs quickly and effectively.

Proving the disappearance of hydrocarbons is important to the success of a bioremediation project. An accurate measurement of hydrocarbons and their biodegradation products is needed to confirm that petroleum was actually consumed by bacteria (discussed in Chapter 7, Section 7.3). One method of confirming biodegradation of petroleum is the coupled measurement of biodegradation rates by proxy methods and the disappearance of the contaminant. Biodegradation rates do not, in and of themselves, prove the decomposition of contaminants. Measurement of biodegradation rates, however, can be an easy way to demonstrate that the potential exists for contaminant removal. While measures of biodegradation rates are often used to estimate time to closure for a site, or proof of technology, biodegradation rates can be unreliable. Common measures of aerobic biodegradation are loss of contaminants, oxygen (O2) consumption, and carbon dioxide (CO2) evolution. Unfortunately, the CO2 can result from non-biological sources (see Chapter 7, Section 7.2.2.2 for additional discussion). Particularly in low pH groundwater, pH adjustment made during bioremediation could result in CO2 off-gassing from groundwater. Oxygen depletion in the subsurface is also not proof of biodegradation.

Introduction

Frozen soil is defined as a soil where the soil moisture has turned totally or partially into ice. On the other hand, permafrost is defined solely on the basis of soil temperature. If the soil temperature remains below 0 °C for at least two years, the soil is considered permafrost. The upper layer of the permafrost undergoes a cyclic temperature change during the year from frozen in the winter to thawed in the summer. This layer is called the active layer or seasonally thawed layer. The active layer in a permafrost region can extend from as little as 20 cm to about 2 m (Shur et al. 2005) depending on climate, soil texture, and organic content above mineral soil. In areas without permafrost the layer of soil which is frozen in the winter is called the seasonally frozen layer. Most permafrost on earth is thousands of years old, but some can be quite new. In permafrost regions, contaminant impacts generally initiate at or near the soil surface and affect the active layer, suprapermafrost water, and uppermost permafrost (Chapter 3). It is this realm that most concerns environmental scientists and engineers tasked with environmental cleanup. A thorough understanding of properties of the active layer and the upper permafrost is necessary for planning and implementing effective remediation of cold media.

Review and recent advances

Thermal and physical properties of frozen ground

Thermal conductivity of soils

The thermal conductivity of soil is the measure of its ability to conduct heat. Soil thermal conductivity is a function of the thermal state of the ground (frozen or unfrozen), water content, dry density, gradation, and mineralogy.

Introduction

It is well established that microbial activity is slower at low temperatures, and that there is a corresponding decrease in biodegradation rates (Paul and Clark 1996; Walworth et al. 1999; Scow 1982; Ferguson et al. 2003b; discussed in Chapter 4). As temperatures fall to near the freezing point of water, biomineralization of hydrocarbons practically ceases. Evaporation rates are also slower at low temperature, although diesel products and more volatile fuels continue to volatilize below 0 °C. For most cold regions, soil is typically unfrozen for only 6–8 weeks, affording a short in situ or passive ex situ treatment season. Even when the ground is thawed, temperatures are generally lower than optimal for hydrocarbon-degrading bacteria (Braddock et al. 2001; Rike et al. 2003).

At their simplest, thermally enhanced bioremediation schemes aim to increase microbial activity by increasing soil temperatures and extending the period when the ground is unfrozen. Modern integrated designs go much further – they typically incorporate some form of venting to promote volatilization, and deliver nutrients, oxygen, and water to hydrocarbon-degrading bacteria in attempts to optimize bioactivity. They are also designed to prevent off-site migration of contaminants and nutrient-enriched waters.

Relative to other remediation options, thermally enhanced bioremediation is a low-cost treatment option (see Chapter 1, Figure 1.1). It is typically much cheaper than bulk extraction and disposal or on-site combustion/desorption treatments, perhaps by a factor of five or more, but approximately two to four times more expensive than landfarming (Chapter 9).

Introduction

Nutrients are required to support biological activity, and hence bioremediation. It is recognized that, although the microbial community requires numerous nutrients, nitrogen and phosphorus are the nutrients most often lacking, and thus limiting to biological hydrocarbon degradation in cold region soils (Mohn and Stewart 2000). Numerous studies have reported that biodegradation of hydrocarbon contaminants in cold region soils has been enhanced by the addition of one or both of these nutrients (Walworth and Reynolds 1995; Braddock et al. 1997; Walworth et al. 1997; Braddock et al. 1999; Mohn and Stewart 2000; Mohn et al. 2001; Ferguson et al. 2003a).

Nitrogen most often provides positive responses, although methodologies for determining application levels are not well defined. Proper nitrogen management can increase cell growth rate (Hoyle et al. 1995), decrease the microbial lag phase (Lewis et al. 1986; Ferguson et al. 2003a), help to maintain populations at high activity levels (Lindstrom et al. 1991), and increase the rate of hydrocarbon degradation (Braddock et al. 1997; Braddock et al. 1999). Whereas many studies indicate positive effects of supplemental nitrogen (Rasiah et al. 1991; Allen-King et al. 1994; Walworth and Reynolds 1995), a surprisingly large number report no benefit, or even deleterious effects when excessive levels of nitrogen are applied (Watts et al. 1982; Brown et al. 1983; Huntjens et al. 1986; Morgan and Watkinson 1990; Genouw et al. 1994; Zhou and Crawford 1995; Braddock et al. 1997; Walworth et al. 1997; Braddock et al. 1999; Mohn et al. 2001; Ferguson et al., 2003a).

Introduction

Bioremediation in cold climates is frequently regarded with skepticism. Owners of polluted sites and regulatory agencies may doubt the effectiveness of biological degradation at near freezing temperatures. While it is true that biodegradation rates decrease with decreasing temperatures, this does not mean that bioremediation is inappropriate for cold regions. Microbial degradation of hydrocarbons occurs even around 0 °C (Chapter 4). In remote alpine, Arctic, and Antarctic locations, excavation and shipping of contaminated soil may be prohibitively expensive. Bioremediation may be the most cost-effective alternative. This chapter discusses microbial adaptation to cold temperatures as well as results of laboratory and field studies of bioremediation at low temperatures.

Microorganisms can grow at temperatures ranging from subzero to more than 100 °C. Microbes are divided into four groups based on the range of temperature at which they can grow. The psychrophiles grows at temperatures below 20 °C, the mesophiles between 20 °C and 44 °C, the thermophiles between 45 °C and 70 °C, and the hyperthermophiles require growth temperatures above 70 °C to over 110 °C. The term “cold-adapted microorganisms” (CAMs) is frequently used for describing bacteria growing at or close to zero degrees Celsius. Depending on the cardinal temperatures (the minimal, the optimal, and the maximum growth temperature), CAMs can be classified as psychrophiles or psychrotrophs. Morita's (1975) definition, which holds that psychrophiles have a maximum growth temperature of less than 20 °C and an optimal growth temperature of less than 15 °C, while psychrotrophs have a maximum temperature of 40 °C and an optimal growth temperature higher than 15 °C, is widely accepted.

Introduction

In this book, current scientific knowledge and practical experiences with bioremediation of petroleum-contaminated soils in cold regions are reviewed and compiled. We now more fully understand the inter-relationships between cold temperatures, soil and water properties, and biological processes. This aids decision making about practical remediation treatment for petroleum-contaminated sites in cold regions. Landfarming and enhanced bioremediation schemes have emerged as viable soil treatment methods that offer a number of advantages over other methods. Nevertheless, work still needs to be done to optimize these methods, and with regards to evaluating phytoremediation and rhizosphere enhancement potentials for cold soils.

Two emerging technologies have been identified that could offer significant cost savings; low-cost heating and controlled-release nutrient systems are described briefly here (see also Chapter 8). In addition, natural attenuation has received little rigorous evaluation for use in cold soils. The main limitation for natural attenuation in cold regions is the low rate of degradation, coupled with off-site migration that can be relatively rapid in soils or gravel pads that have a poor adsorption capacity. Permeable reactive barriers are one groundwater treatment technology that could buy time for slower in situ techniques such as natural attenuation to take place. An outline of emerging permeable-reactive barrier technology is presented here, although full-scale trials are not yet complete. It is possible that such in situ techniques, when coupled with aeration, sparging and biostimulation could offer methods for groundwater treatment in cold regions.

Introduction

Landfarming has been described as “a simple technique in which contaminated soil is excavated and spread over a prepared bed and periodically tilled until pollutants are degraded” (Vidali 2001) but, in practice, it can be either an ex situ or in situ technique. Landfarming generally uses a combination of volatilization and biodegradation to reduce hydrocarbon concentrations. For biodegradation to be effective, stimulating aerobic soil microorganisms is essential; this is commonly accomplished by adding nutrients and mixing the soil to increase aeration. Aerating the soil in this way also increases the loss of hydrocarbon contaminants to the atmosphere via volatilization. Volatilization of diesel and lighter hydrocarbons greatly assists the remediation process but it is less effective for heavier molecular weight hydrocarbons such as crude oil.

For in situ landfarming it is possible to treat only relatively shallow layers of soil where reasonable oxygenation can be maintained. In ex situ landfarming, excavated contaminated soil is spread as a thin layer in a treatment bed that is often lined with an impermeable layer to control leaching and runoff. Ex situ landfarming can be as simple as soil spread in a cleared area or it can be a major construction with contouring or drainage systems or both for removal of excess water. Plumbing can also be used for the application of water, either alone or in combination with nutrients or other amendments, to the landfarm surface.

Introduction

The miRBase database (formerly entitled the microRNA Registry) is the primary online repository for microRNA (miRNA) sequences and annotation (Griffiths-Jones, 2004; Griffiths-Jones et al., 2006). When laboratories first began to clone and sequence increasing numbers of miRNAs (Lagos-Quintana et al., 2002) it became apparent that a single resource for the naming, annotation and dissemination of published miRNAs was urgently required. With no predefined nomenclature or central repository for miRNA sequences there was a distinct danger that these sequences would appear in journals with inconsistent names. Furthermore, if miRNAs were independently identified by multiple laboratories and had multiple ambiguous identifiers this could hamper subsequent analysis. Previously, the RFAM project (Griffiths-Jones et al., 2003) at the Wellcome Trust Sanger Institute had been cataloging and identifying RNAs and their evolutionary relationships and hence already had much of the expertise required. A number of laboratories involved in miRNA research discussed these issues and published a collaborative document detailing the agreed nomenclature for miRNAs and announcing the miRNA registry as their main repository (Ambros et al., 2003).

Recently the resource has expanded to include miRNA annotations and automatically predicted targets for animal miRNAs and has been renamed miRBase (Griffiths-Jones et al., 2006). This resource currently attracts a very large number of visitors and registered over 1.5 million page hits in July 2006 alone, illustrating the growing scientific interest in these important regulatory molecules.

As detailed in this volume, the tiny RNAs known as microRNA molecules regulate several biological processes, such as development, differentiation and disease biology. However, how they do so has remained unclear. This book does not cover the recent reports on their involvement in the immune system and even stress responses in the heart. It is becoming clear that certain microRNAs are emerging as key players in stage-specific expression in the immune system. Almost two decades ago, biologists began to identify the roles of genes by knocking them out and studying these “knock-out” animals, which lacked the proteins encoded by the targeted genes. Now, by using the same strategy it is possible to remove portions of genes that make scraps of RNA. Overall, studies of knock-out mice so far have helped us to understand how genes govern health and disease. As the field of microRNomics is rapidly growing at such a rapid pace, the material contributed in this volume might already be slightly outdated. In fact by the time we finalized the book chapters, the roles of microRNAs in immunology, cardiology, diabetes and unicellular organisms had been reported. I will therefore review these topics here.

MicroRNAs in immunology

Over 30% of our genes are under the control of small molecules called microRNAs. They prevent specific genes from being turned into protein and regulate many crucial processes such as cell division and development.

Introduction

Small, non-coding RNAs of 18–32 nucleotides have emerged as evolutionarily conserved potent regulators of gene expression in the past decade. Studies of the function of small RNAs demonstrated their pivotal roles in various aspects of cell and developmental biology, as detailed in the previous chapters of this book. As the newest citizens of the small RNA world, Piwi-interacting RNAs (piRNAs) of mostly 26–32 nucleotides in length were discovered in 2006 in mammalian testes (Aravin et al., 2006; Girard et al., 2006; Grivna et al., 2006a; Lau et al., 2006; Watanabe et al., 2006). They are so named because they interact with the Piwi sub-family proteins of the evolutionary conserved Argonaute/Piwi protein family. piRNAs also exist in large numbers in fly (Brennecke et al., 2007; Gunawardane et al., 2007) and fish gonads (Houwing et al., 2007), implying the evolutionary conservation of their function. They differ from miRNAs and siRNAs in size, biogenesis, expression pattern, and possibly function. Although still remaining to be fully elucidated, clues about their biogenesis and function have started emerging. There are over 60 000 different species of piRNA identified so far, much exceeding the several hundreds of miRNAs that have been discovered. This fascinating complexity of piRNAs provides unprecedented opportunities for unraveling novel and diverse mechanisms of small RNA-mediated gene regulation. This chapter will summarize the latest progress on piRNAs.

Ago/Piwi protein family comprises two sub-families

Any description of small RNAs would be incomplete without an account of their protein partners: the Argonaute/Piwi (Ago/Piwi) family proteins.