Summary

The constitutive fibrils of the Lewy body contain all three neurofilament subunits in an altered form. Our recent in situ hybridization studies of the low molecular weight subunit of the neurofilament suggest that altered neurofilament expression is not implicated in the formation of the Lewy body. Using the Mallory body of the liver as a dynamic model of intermediate filament inclusion formation, we also demonstrated that the filaments are first normally assembled and modified in a subsequent step. We propose a speculative model of Lewy body pathogenesis in which neurofilaments are assembled normally and subsequently undergo phosphorylation, proteolysis and crosslinking at a post-translational/post-assembly stage.

Introduction

The Lewy body (LB) is a major pathological feature of Lewy body disorders including idiopathic Parkinson's disease (PD) and diffuse Lewy body disease (DLB). As we discussed in our recent review (Pollanen et al., 1993), direct and indirect analysis of the LB has shown that its constitutive fibrils contain all three neurofilament (NF) subunits of molecular weights 68 kD (NF-L), 150 kD (NF-M) and 200 kD (NF-H) in both phosphorylated and nonphosphorylated forms. The NF epitopes are spatially distributed with a predominance of NF-L or amino-termini of NF subunits in the central portion of the inclusion, a phenomenon which suggests a possible segregation of proteolytic fragments. The presence of ubiquitin, ubiquitin-C-terminal hydrolase and ingestin (multicatalytic proteinase or proteasome) in the LB also suggests that proteolysis is implicated in its pathogenesis. Finally, the detergent-insolubility of these fibrils indicates that the NF proteins which form the LB are further altered and probably crosslinked. Taken together, these studies suggest that altered phosphorylation/dephosphorylation and proteolysis of NF are key post-translational events in LB pathogenesis.

Introduction

Explosives and related compounds have become widely recognized as serious environmental contaminants. Among the nitrosubstituted aromatic compounds causing particular concern are 2, 4, 6-trinitrotoluene (TNT), 2, 4, 6-trinitrophenol (picric acid), and many nitro- and/or amino-substituted aromatics that result from the manufacture and transformation of explosives. The threat posed by the presence of these compounds in soil and water is the result of their toxicity and is compounded by their recalcitrance to biodegradation.

Contamination by nitroaromatic compounds, especially TNT, stems primarily from military activities (Boopathy et al., 1994). During the manufacture of explosives and the disposal of old munitions, large quantities of water became contaminated. This wash water was typically disposed of in unlined lagoons that facilitated the slow release of the explosives from the soil in the lagoons into ground water, lakes, and rivers.

The mutagenicity of TNT (Kaplan & Kaplan, 1982a; Won et al., 1976), as well as its toxic effects on algae and fish, humans, and other vertebrates, make it an environmental hazard (Hudock & Gring, 1970; Smock et al., 1976; Won et al., 1976). TNT has been listed as a priority pollutant by the U.S. Environmental Protection Agency (Keither & Telliard, 1979). Tan et al. (1992) compared the mutagenicity of the biologically reduced forms of TNT (2-amino-4, 6-dinitro toluene and 4-amino-2, 6-dinitrotoluene) to TNT, hexahydro-l, 3, 5-trinitro-l, 3, 5-triazine (RDX), octahydro-l, 3, 5, 7-tetranitro-l, 3, 5, 7-tetrazocine(HMX), and N-methyl-N, 2, 4, 6-tetranitroaniline (Tetryl) using the Salmonella/mammalian microsome plate incorporation method. They found that the reduction of the nitro groups of TNT to the amino products caused a decrease in the mutagenic activity of the compounds proportional to the number of amino groups formed.

Bioremediation is not a new concept in the field of applied microbiology. Microorganisms have been used to remove organic matter and toxic chemicals from domestic and manufacturing waste effluents for many years. What is new, over the past few decades, is the emergence and expansion of bioremediation as an industry, and its acceptance as an effective, economically viable alternative for cleaning soils, surface water, and groundwater contaminated with a wide range of toxic, often recalcitrant, chemicals. Bioremediation is becoming the technology of choice for the remediation of many contaminated environments, particularly sites contaminated with petroleum hydrocarbons.

Bioremediation has also become an intensive area for research and development in academia, government, and industry. Partly because of new laws requiring stricter protection of the environment and mandating the cleanup of contaminated sites, funding for both basic and applied research on bioremediation by government agencies, as well as by private industry, has increased dramatically over the past decade. As a result, rapid progress has been made in developing effective, economical microbial bioremediation processes. In an even broader sense, this increased activity has led to a surge of interest in ‘environmental microbiology’, a field covering a spectrum of disciplines, including microbial physiology and ecology, molecular genetics, organic chemistry, biochemistry, soil and water chemistry, geology, hydrology, and engineering. In the academic research environment, bioremediation has, in effect, become so scientifically broad and complex in both its basic and applied aspects that it has of necessity evolved into a multidisciplinary field that requires a ‘research center’ approach. At the University of Idaho, as just one example, we have built our multidisciplinary environmental remediation research program within the University of Idaho Center for Hazardous Waste Remediation Research.

Metals in the environment

Metal pollution is a widespread problem; in fact, in industrially developed countries it is normal to find elevated levels of metal ions in the environment. In addition, it has been estimated that approximately 37% of sites in the U.S. contaminated with organic pollutants, such as pesticides, are additionally polluted with metals (Kovalick, 1991). Despite this, biological treatment or bioremediation of contaminated sites has largely focused on the removal of organic compounds, and only recently has attention turned to the treatment of metal-contaminated wastes (Brierley, 1990; Summers, 1992). Due to their toxic nature, the presence of metals in organic-contaminated sites often complicates and limits the bioremediation process. Such metals include the highly toxic cations of mercury and lead, but many other metals are also of concern, including arsenic, beryllium, boron, cadmium, chromium, copper, nickel, manganese, selenium, silver, tin and zinc.

Metals are ubiquitous in nature and even those metals generally considered as pollutants are found in trace concentrations in the environment (see Table 10.1). For the most part, metal pollution problems arise when human activity either disrupts normal biogeochemical cycles or concentrates metals (see Table 10.2). Examples of such activities include mining and ore refinement, nuclear processing, and industrial manufacture of a variety of products including batteries, metal alloys, electrical components, paints, preservatives, and insecticides (Gadd, 1986; Hughes & Poole, 1989; Suzuki, Fukagawa & Takama, 1992). Metals in these products or metal wastes from manufacturing processes can exist as individual metals or more often as metal mixtures.

Introduction

Polychlorinated biphenyls (PCBs) are a group of related chemicals (congeners) which were produced during the middle of this century as chlorinated derivatives of biphenyl. There are 209 congeners distinguished by the number and position of chlorine atoms on a biphenyl backbone, although many of these congeners are not found in the commercial PCB mixtures (marketed under the tradename ‘Aroclor’ in the U.S., ‘Clophen’ in Europe, and ‘Kaneclor’ in Japan) (Ballschmiter & Zell, 1980; Hutzinger et al., 1983; Erickson, 1992). The chemical and thermal stability of these compounds formed the basis for the widespread use of PCBs as the fire-retardant fluid in electrical equipment, heat exchangers, hydraulic fluids, and compressor fluids. However, this chemical stability also carried with it a degree of biological stability that has resulted in the accumulation of PCBs at sites of its manufacture, use, storage, and disposal.

Although the term PCBs has evoked the specter of the grim reaper, at the outset of this review it should be stated that this visceral response to PCBs has at times bordered on the irrational. As our society moves into the next century and we increasingly address our environmental problems, we must take a realistic view of the true risks posed by each of the chemical targets that we address. It should also be remembered that in their time PCBs were an important life-saving invention. Prior to 1929 electrical equipment contained flammable fluids which often ignited, creating conflagrations that truly posed serious risks to human health. The use of chlorinated organics, including PCBs, as fire-retardant liquids helped create a safer environment for our growing industrial society.

Introduction to the soil system

One of the major obstacles in bioremediation of soils contaminated with synthetic organic compounds is the failure of laboratory remediation schemes to simulate the impact of field soil conditions on both the contaminant and the microorganism (Rao et al., 1993). The purpose of this chapter is to introduce those topics which must be considered in order to develop an effective bioremediation strategy for soils contaminated with organic pollutants. My emphasis is on providing a comprehensive overview of the complexity of the soil system as it relates to bioremediation.

The soil environment is a dynamic one which includes gas, liquid, and solid phases. It is imperative in any soil bioremediation process to have a clear understanding of these phases and how they interact. This includes not only the chemical characteristics of soil colloids, but the physical arrangement of components. The first section is meant as a brief introduction to soil chemical and physical properties and is intended primarily for those unfamiliar with the area.

The inorganic solid phase in soil

The solid phase consists of both inorganic and organic components, which in many cases form a heterogeneous mixture defined as organo-mineral complexes. In-depth treatments of soil chemistry, humus chemistry, and mineralogy are available (Aiken et al., 1982; Bohn et al., 1985; Dixon and Weed, 1989; Sposito, 1989; McBride, 1994; Stevenson, 1994). Soil inorganic components include both crystalline materials in the form of layer silicates as well as more poorly crystalline oxides, hydroxides, and oxyhydroxides (collectively termed hydrous oxides).

Chlorophenols are common environmental contaminants originating mainly from their use as wide-spectrum biocides in industry and agriculture, their formation during pulp bleaching and the incineration of organic material in the presence of chloride. Chlorophenol use as biocides has resulted in soil and groundwater contamination; pulp bleacheries discharge wastewaters into aquatic environments; and combustion results in the contamination of all environmental compartments. Chlorophenols have the potential of being biotransformed and/or mineralized in both aerobic and anaerobic systems and degradation pathways have been carefully delineated. In aquatic and terrestrial environments chlorophenols become bound to humic materials, affecting their bioavailability, and possibly offering a useful method of detoxification. Although chlorophenols persist in many environments, because of inappropriate conditions for biodegradation, biological methods have been utilized in groundwater and soil remediation of chlorophenols. Composting and landfarming are the most common soil remediation methods, while on-site bioreactors are used for groundwater remediation. In situ bioremediation for restoration of contaminated subsurface environments has gained significant interest and the first full-scale operations are in progress. The fate of chlorophenol contaminants in aquatic sediments has been widely studied and the results serve as a basis of future efforts in remediation of sediments.

Sources of contamination

The forest industry has used extensive amounts of chloropheols (CPs) for the preservation of timber against blue sapstain fungi. In the U.S., pentachorophenol (PCP) ia mainly used (Cirelli, 1978), while in Europe and Japan, various CP congener mixtures are typical. Preservative solutions are prepared either by dissolution of CPs in sodium hydroxide to produce concentrated chlorophenate solutions, or dissolution in fuel oil or kerosene. If these solvents are not available, liquid petroleum gas, methylene chloride, isopropyl alcohol, or methanol are used.

Introduction

Hydrocarbon degradation by microorganisms is a natural manifestation of the carbon cycle. Only when hydrocarbon pollutants occur in excessively high concentrations, or prove highly toxic or are recalcitrant to existing bacterial degradative processes do they become the focus of remediation efforts. Intrinsic (or natural) bioremediation therefore defines the fate of most environmental chemicals, whether natural or anthropogenic. Indeed, through chemical modifications such as halogenation, a great deal of effort has gone into creating classes of hydrocarbons, such as the polychlorinated biphenyls (PCBs), that are more environmentally stable than their unmodified analogs. The recalcitrance of these compounds has led us to attempt to determine why the organisms normally responsible for carbon cycling fail when challenged by certain pollutants. Through the process of genetic engineering, strain development, enzyme redesign and pathway construction we can then undertake the molecular manipulation of biodegradative systems to improve their utility in bioremediation.

Biodegradation depends on the microbial production of enzymes capable of catalyzing chemical reactions that will transform (or, ideally, mineralize) pollutants. Bioremediation technologies can be enhanced in several ways, but the existing biochemical capabilities of the organisms should be reviewed before any anticipated genetic alterations are considered.

The most frequent means of stimulating bioremediation include:

1. Chemical modifications of the environment (Nelson et al., 1986; Mueller et al., 1991a, b, 1992; Pritchard & Costa, 1991; McCarty & Semprini, 1994) can include the addition of chemicals which act as electron acceptors or supplemental electron donors, or which enhance bioavailability (Rosenberg et al., 1979; Georgiou, Lin & Sharma, 1992).

2. Physical modifications of polluted sites may relate to ex situ or in situ treatments.

Chlorinated aliphatic compounds in the environment

Natural products and synthetic compounds

Chlorinated aliphatic compounds are abundant in nature. Among the more than 1500 natural product organohalides that have been identified (Gribble, 1992) are a significant number of chlorinated aliphatic compounds. Quantitatively, chloromethane is the most significant. It is estimated that 5 × 109kg are produced annually, principally by soil fungi (Rasmussen, Khalil & Dalluge, 1980). The biochemical reaction producing chloromethane has been investigated (Wuosman & Hager, 1990) and soil bacteria have been identified that grow on chloromethane as a sole carbon and energy source (Hartmans et al., 1986; Traunecker, Preub & Dieckert, 1991). Hence, chloromethane is one of the almost innumerable intermediates in the global carbon cycle.

Chlorinated aliphatic compounds of industrial origin are perhaps more widely known. They include chloroalkanes, chloroalkenes, and chlorinated cycloaliphatic compounds (Figure 9.1). The principal usages of these compounds are as solvents and synthetic intermediates. 1, 2-Dichloroethane is one of the most heavily used commodity chemicals, with 17.95 billion pounds produced in the United States in 1993 (Reisch, 1994). Industrial solvents such as dichloromethane, trichloroethylene (TCE), and tetrachloroethylene (PCE) have been valuable because of their relative chemical inertness, ease of evaporative transfer, and relatively low mammalian toxicity. Other prominent solvents are 1, 1, 1-trichloroethane, chloroform, and carbon tetrachloride. The latter two have seen greatly decreased usage due to demonstrated toxicity and carcinogenicity (Anders & Pohl, 1985). Chlorofluorocarbons used as refrigerants are predominantly C1 and C2 alkanes. Although they show excellent heat transfer and chemical inertness characteristics, their well-documented ozone-depleting effects are leading to drastic constraints on their use (Molina & Rowland, 1974).

Introduction

The selection of microbiological processes for treating soils and groundwater contaminated with organic pollutants requires characterization of the waste, selection of an appropriate microorganism or consortium, and information about degradation pathway and rates. This chapter will focus on ex situ processes and the selection of reactors for the treatment of soils and groundwater.

Bioremediation must compete economically and functionally with alternate remediation technologies, which are often incineration and chemical treatments. Bioremediation usually competes well on a cost basis, especially with petroleum products and many solvents. A drawback, however, is the large amount of preliminary information necessary to support process design. When information on waste characteristics, microbial physiology, and the complex options for process design and operation is lacking, bioremediation can be more difficult to apply than alternate technologies. The variable end results of bioremediation are due in large part to these complexities of process design.

The engineering of bioremediation processes relies on information about the site and about candidate microorganisms. Process analysis usually begins with fixed waste characteristics but with options for microbial cultures, reactor types, waste pretreatment, and process operating conditions. Laboratory measurements are necessary to explore these options and to design an efficient process. Laboratory tests that examine degradation rates as functions of critical operating parameters such as pH, oxygen and nutrient concentrations, microbial composition, soil particle size, temperature, and redox potential shape the design of a bench-scale process. At a small scale mass transfer effects such as agitation and aeration are also explored. These tests form the basis for scale-up to the field scale and for the implementation of process control.

Many aspects of this design strategy have been presented.

Introduction

During this century the demand for petroleum as a source of energy and as primary raw material for the chemical industry has resulted in an increase in world production to about 3500 million metric tons per year (Energy Information Administration, 1992). It has been estimated that approximately 0.1%, 35 million tons, enters the sea per annum (National Research Council, 1985). A great part of the oil pollution problem results from the fact that the major oil-producing countries are not the major oil consumers. It follows that massive movements of petroleum have to be made from areas of high production to those of high consumption. Although the large crude oil spills following tanker accidents (e.g., Table 4.1) receive the most public attention, tanker accidents represent only a small fraction, about one million tons, of the total input. By comparison, oil input to the sea from natural sources, principally seeps, is about 0.5 million tons annually. The major sources of oil pollution are municipal and industrial wastes and runoffs, leaks in pipelines and storage tanks, and discharge of dirty ballast and bilge waters. Since the residence time of hydrocarbons in the sea is decades or centuries (Button et al., 1992), dissolved hydrocarbons circulate in the deep oceans, thereby constituting a global problem.

The toxicity of petroleum hydrocarbons to microorganisms, plants, animals and humans is well established. In fact, many bioassays for petroleum pollution have been developed which depend on low concentrations (5–100mg/l) of crude oil or petroleum fractions killing or inhibiting the growth of microalgae and juvenile forms of marine animals.

Most organic chemicals and many inorganic ones are subject to enzymatic attack through the activities of living organisms. Most of modern society's environmental pollutants are included among these chemicals, and the actions of enzymes on them are usually lumped under the term biodegradation. However, biodegradation can encompass many processes with drastically differing outcomes and consequences. For example, a xenobiotic pollutant might be mineralized, that is, converted to completely oxidized products like carbon dioxide, transformed to another compound that may be toxic or nontoxic, accumulated within an organism, or polymerized or otherwise bound to natural materials in soils, sediments, or waters. More than one of these processes may occur for a single pollutant at the same time. The chapters in this book will discuss these phenomena in relation to specific groups of xenobiotic pollutants, since these processes ultimately determine the success or failure of bioremediation technologies.

Bioremediation refers to the productive use of biodegradative processes to remove or detoxify pollutants that have found their way into the environment and threaten public health, usually as contaminants of soil, water, or sediments. Though biodegradation of wastes is a centuries-old technology, it is only in recent decades that serious attempts have been made to harness nature's biodegradative capabilities with the goal of large-scale technological applications for effective and affordable environmental restoration. This development has required a combination of basic laboratory research to identify and characterize promising biological processes, pilot-scale development and testing of new bioremediation technologies, their acceptance by regulators and the public, and, ultimately, field application of these processes to confirm that they are effective, safe, and predictable.

Introduction

Fossil energy reserves are valuable natural resources that underpin most major world economies. The extraction, transport and utilization of these resources inevitably leads to the release of these substances to environmental compartments where they are deemed undesirable. For instance, petroleum or petroleum distillation products often occur as contaminants in soils, aquifers and surface waters via a myriad of mechanisms including leaking underground storage tanks, aboveground spills and release by marine transport vessels. Since environmental matrices (air, water and soil) are completely integrated, all are susceptible to a reduction in quality and often quantity due to the release of pollutant materials. When this occurs, a great deal of concern is associated with the impact of the hydrocarbons on humans and recipient environments. However, this impact cannot be correctly gauged without information on the transport and fate characteristics of the individual contaminants.

A major factor governing the transport and fate of contaminant hydrocarbons is their susceptibility to metabolism by aerobic and anaerobic microorganisms. While a plethora of information is available on the prospects for aerobic biodegradation, comparatively little is understood about anaerobic hydrocarbon biotransformation. However, it is well recognized that anaerobic microbial activities directly or indirectly impact all major environmental compartments. In many environments, most notably the terrestrial subsurface, oxygen concentrations are often initially low. With rapid utilization by hydrocarbonoclastic microorganisms and limited rates of reoxygenation, oxygen becomes depleted. Without the reactive power of molecular oxygen, the biodegradation rate of hydrocarbons slows down and contamination problems are exacerbated. Nevertheless, some of the most toxicologically important components of petroleum can be metabolized, even in the absence of oxygen.