Chlorinated solvents are some of the most frequently occurring types of contaminants in soil and groundwater at designated Superfund and other hazardous waste sites in the United States. They are organic compounds that contain chlorine atoms and their properties make them ideal for many industrial-cleaning applications such as degreasing oils and fats. Common solvents include tetrachloroethene (PCE) and trichloroethene (TCE), used extensively in the dry-cleaning industry, and 1,1,1-trichloroethane (TCA) and Methylene Chloride typically used as industrial degreasers.
As shown in FIG. 1, when released into the subsurface, chlorinated solvents tend to sink through the saturated zone as they are denser than water. As a result, small droplets (ganglia) get trapped in the soil ‘pore-space’ as a non-aqueous phase liquid (NAPL), which can act as a long-term source of dissolved phase contamination. These NAPL source zones can hamper any site remediation effort as they are difficult to treat and detect.
Anaerobic reductive dechlorination is one treatment process that has been successfully used to remediate soil and groundwater contaminated with chlorinated solvents. The occurrence of different types and concentrations of electron donors such as native organic matter and electron acceptors such as oxygen and chlorinated solvents determine which reductive dechlorination occurs during the natural attenuation of a site.
Reductive dechlorination only occurs in the absence of oxygen; chlorinated solvents substitute for oxygen in the physiology of the microorganisms carrying out the process. Remedial treatment technologies usually introduce an oxygen scavenger to the subsurface to ensure this process occurs immediately.
Anaerobic conditions occur when anaerobic bacteria use the chlorinated contaminants as the electron donors and, in most instances, allow the microorganism to derive useful amounts of energy from the reaction. It has been shown that vinyl chloride can be oxidized to carbon dioxide, water, and chloride ion via Fe (III) reduction. Significant anaerobic mineralization of dichloroethene, vinyl chloride, and methylene chloride also has been reported in the literature.
Halorespiration is a type of anaerobic respiration in which a chlorinated compound is used as a terminal electron acceptor. In this reductive dechlorination process, which enables the conservation of energy via electron transport phosphorylation, one or more chlorine atoms are removed and replaced by hydrogen. Halorespiration, also referred to as dehalorespiration, occurs when the organic compound acts as an electron acceptor (primary growth substrate) during reductive dechlorination. During dehalorespiration, the chlorinated organic compounds are used directly by microorganisms (termed “dehalorespirators”), such as an electron acceptor while dissolved hydrogen serves as an electron donor:H2+C—Cl→C—H+H++Cl−where C—Cl represents the chlorine bond to the carbon in the chlorinated ethene molecule. Dehalorespiration requires not only the presence of competent microorganisms, but also the appropriate quantity and quality of electron donors which serve as the driving force for dehalorespiration. A variety of electron donors have been shown to sustain reductive dechlorination; however, it has only recently been recognized that dissolved hydrogen is the actual electron donor in dehalorespiration (Wang, 2000).
Dehalorespiration occurs as a two-step process which results in the interspecies hydrogen transfer by two distinct strains of bacteria. In the first step, bacteria ferment organic compounds to produce hydrogen. During primary or secondary fermentation, the organic compounds are transformed to compounds such as acetate, water, carbon dioxide, and dissolved hydrogen. Fermentation substrates are either biodegradable nonchlorinated contaminants, or naturally occurring organic carbon. In the second step, the nonfermenting microbial consortia utilize the hydrogen produced by fermentation for dehalorespiration. Although compounds produced during fermentation have been demonstrated to drive dehalorespiration, hydrogen appears to be the most important electron donor for this process.
Dehalorespiration requires sufficient substrates to establish and maintain anaerobic conditions conducive to reductive dechlorination for a period of time and to degrade all unwanted constituents and their daughter products. Common substrates used include acetate, propionate, butyrate, benzoate, glucose, lactate, formate, methanol, toluene, molasses, cheese whey, corn steep liquor, corn oil, hydrogenated cottonseed oil beads, solid food shortening, beef tallow, melted corn oil margarine, coconut oil, soybean oil, and hydrogenated soybean (Sieczkowski, 2012). These compounds serve as the precursors to dissolved hydrogen generation via fermentation. Obligate proton reducers are required to ferment organic substrate present in the subsurface environment to waste products of acetate, formate, dissolved hydrogen, and carbon dioxide (Zehnder, 1988). After fermentation, dissolved hydrogen becomes available for subsequent use by other microorganisms, such as methanogens and dehalorespirators. This syntrophic relationship of hydrogen producers and consumers is known as interspecies hydrogen transfer. Dehalorespiration relies on the presence of fermentable organic substrates that produce dissolved hydrogen.
In addition to the quality of an electron donor, quantity must also be addressed. Since the dissolved hydrogen produced from the fermentation of organic substrates can be used by a variety of microorganisms (e.g. methanogens and dehalorespirators), it is important to consider the competition amongst these microorganisms for dissolved hydrogen when assessing the potential for dehalorespiration (Gossett and Zinder, 1997). Researchers have used the Monod model to examine the uptake of dissolved hydrogen by competing bacteria groups. The Monod model is based on microbial growth under a limiting substrate (e.g. dissolved hydrogen) and is expressed as:
  μ  =            μ      max        ⁢          S                        K          s                +        S            where μ is the specific growth rate, μmax is the maximum specific growth rate, S is the substrate concentration, and Ks is the half-saturation constant. The parameter Ks gives an indication of how rapidly μ approaches μmax. A lower Ks suggests that a microorganism will reach its maximum specific growth rate at a lower substrate concentration than another microorganism with a higher Ks, and hence are better scavengers when competing for the same limiting substrate.
Smatlak and Gossett (1996) compared the kinetics of dissolved hydrogen use by methanogens and dehalorespirators and obtained Monod-half saturation constants, Ks, of approximately 1.0 and 0.1 mM H2 for methanogens and dehalorespirators, respectively. Their results suggest that dehalorespirators are better scavengers for dissolved hydrogen than methanogens, and that the choice of an electron donor that ferments to release dissolved hydrogen at slow, steady, and low levels, such as propionate or butyrate, would favor dehalorespirators over methanogens in the competition for hydrogen (Wang, 2000).
In addition to electron donors, deficiencies of available vitamins and nutrients can also limit dehalorespiration; such nutrients may include organic carbon, nitrogen, phosphorous, amino acids, trace elements, and vitamin B12. The complexity of undefined microbial communities makes the understanding of specific nutritional requirements difficult. Yeast extract, a complex substrate, has been shown to increase dechlorination rates to those greater than of simpler substrates. Nutrient amendments to a contaminated aquifer may also benefit reductive dechlorination by stimulating the activity of non-dehalorespirators, which for example, prevent the accumulation of an inhibitory product (Mohn and Tiedje, 1992). Maymo-Gatell et al. (1995) investigated the nutritional requirements of an anaerobic enrichment culture competent at transforming PCE to ethene. Their results suggested that the dehalorespiring culture was dependent on other microorganisms to satisfy some nutritional requirements, and that yeast extract and vitamin B12 play roles in dechlorination activity. Vitamin B12 was also shown to be a factor in sustaining dehalorespiration by Dehalospirillum multivorans (Neumann et al, 1994). Smatlak and Gossett (1996) measured Ks (H2) values of 100 nM for dehalorespirators and 1,000 nM for methanogens, and suggested that dehalorespirators would out-compete methanogens for electron donors only at low dissolved hydrogen concentrations. This implies that reductive dechlorination by dehalorespirators will be optimal when the amount of available electron donor is low, in order to minimize the direction of electron donors to methanogenesis.
In natural systems, including contaminated aquifers, most H2 becomes available to hydrogenotrophic microorganisms through the fermentation of more complex substrates by other members of the microbial consortium. The dechlorinators must then compete with other organisms, such as methanogens and sulfate-reducing bacteria, for the evolved H2 as best shown in FIG. 2. FIG. 3 also describes the distribution of electrons during the microbial breakdown of organic electron donor substrates (Suthersan, 2001).
Therefore, there is a need in the art for an anaerobic reductive decholorination process that controls the release rate of hydrogen that acts as an electron donor in dehalorespiration through use of encapsulated substrates.