Microorganismsand Applications

Due to inherent difficulties in achieving the sterile conditions necessary for prolonged maintenance of pure cultures in field-scale bioreactors, most gas treatment bioreactors contain a mixed culture or consortium of microorganisms. These consortia may be derived from a number of different inocula, from common sources such as sewage-treatment facilities, biofilms from established bioreactors used for similar applications, or soils and waters from areas contaminated with the substrate of interest. In some instances, it is possible to enrich for the desired microbial consortium directly from the bioreactor bed medium, particularly when media components such as soil, compost, peat, or bark chips are used, because these materials naturally contain a mixture of microbes with wide-ranging physiological capacities. Regardless of the inoculum source, the bioreactor must be operated under conditions that select for and maintain, over the life of the bioreactor, microorganisms with the physiological capabilities necessary to catalyze the desired bioconversion. Typical selective factors include the electron donor (i.e., the metaboliz-able energy source), available terminal electron acceptors, supplemental nutrients, pH, Eh, and temperature.

Regardless of the selection strategy used to achieve a microbial consortium with the desired physiological properties, the use of a consortium versus the attempted use of a pure culture of microbes has a number of practical advantages. As mentioned above, it is difficult to achieve the sterility standards necessary to maintain a pure culture of microbes in a gas treatment bioreactor under field conditions. In addition, a pure culture of microbes is often met-abolically incapable of fully degrading a contaminant, so hazardous intermediates may be created and build up within the bioreactor or in effluent streams from the bio-reactor. Because mixed consortia inherently tend to be composed of a number of different microbial species with a gamut of physiological capabilities, undesired intermediates that may be created by pure cultures are often fully degraded by consortia. Similarly, in many applications, the concentrations and chemical compositions of gas streams to be treated may be transient. Consortia generally have a higher probability than pure cultures of adapting to this transience. The contaminant gases and vapors that can be successfully biodegraded can be classified by their role in microbial metabolism.

Electron Donors

Microorganisms require a constant supply of metabolic energy, which is normally derived from the oxidation of either organic or inorganic compounds, with these compounds serving as electron donors to the microorganisms. Many environmentally or industrially significant gases and vapors are metabolically oxidizable by microorganisms and can serve as electron donors. Thus, many different types of gas and vapor streams can be and have been, treated microbiologically (1). Microbially oxidizable gases and vapors that have been treated in bioreactors include hydrocarbons, ketones, ammonia, xylene, alcohol, terpene, and carbon disulfide vapors, as well as gases such as methane and hydrogen sulfide (2-10). In the case of carbon-containing substrates (hydrocarbons, carbon monoxide, etc.), the metabolism of these compounds provides hetero-trophic microbes not only with an electron source, but also with a source of cellular carbon. Non-carbon-containing substrates such as ammonium, while providing a metabolic electron source, must be metabolized by autotrophic microorganisms capable of obtaining their cellular carbon via the fixation of atmospheric carbon dioxide.

Cometabolism

Some substrates cannot serve as sole energy sources for microorganisms, but are nevertheless potentially biocon-vertable. Their biodegradation is achieved in the presence of another compound that serves as the microorganism metabolic electron donor, a process that is termed co-metabolism. Trichloroethylene (TCE) provides a well-documented example of a substrate that is oxidized co-metabolically by microorganisms. Typical of cometabolic TCE degraders are the methanotrophic bacteria, aerobes that oxidize methane as their sole carbon and energy source (11). This oxidation takes place in a sequential manner, with methane first being converted to methanol, which is in turn oxidized to formaldehyde, formate, and finally, carbon dioxide. Along this oxidative pathway, the meth-anotrophs generate cellular energy as well as fixing methane-derived carbon into additional biomass. The first step, in which methanol is formed from methane, is catalyzed by the enzyme methane monooxygenase (MMO) (12,13). Although MMO is selective for its natural substrate, it can, under certain conditions, catalyze the oxidation of a variety of other compounds (12-14), including partially chlorinated aliphatic solvents such as TCE. Two distinct forms of MMO have been identified, membrane MMO (mMMO) and soluble (sMMO) (15,16). sMMO, ex pressed by methanotrophs growing under copper-limited conditions, supports much higher rates of TCE oxidation than does mMMO, which is expressed under conditions of copper sufficiency (17).

Air streams contaminated with TCE and similar chlorinated solvents are generated by industrial operations and the remediation of contaminated ground and water by air stripping. They can be treated in a bioreactor that is started up with methane as the sole carbon and energy source, while supplying the necessary oxygen and supplemental nutrients under copper-limiting conditions (6). After the methanotrophic consortium is established, the methane load to the reactor can be decreased or fed on an intermittent basis concurrently with TCE vapors. The methanotrophic microbes then will be maintained by the lessened methane feed, while catalyzing the oxidation of the TCE via their sMMO. The reduced or intermittent methane feed not only reduces the amount of methane that must be supplied, but is essential in achieving optimal TCE removal rates since methane itself is an effective competitive inhibitor of TCE oxidation. An alternate, but more expensive, strategy used to avoid this type of competitive inhibition is to supply a catabolic intermediate such as formate in order to provide energy to the methanotrophs, because formate does not compete with TCE for access to the sMMO. It should be noted that a number of other microbial oxygenases have been shown to be capable of catalyzing cometabolic TCE oxidation. These include toluene oxygenases from a number of Pseudomonas spp., propane mono-oxygenases from propanotrophs, and ammonia monooxy-genase from Nitrosomonas europea (18-21).

Electron Acceptors

Most gas treatment bioreactors remove contaminants from air streams and oxidize them to innocuous end products (CO2, Cl", etc.), using the oxygen in the air as the electron acceptor. However, there are also gas streams containing little or no oxygen where the objective is to remove a compound that can act as an alternative physiological terminal electron acceptor. This approach has been shown to work for compounds like sulfur oxides (SOA) and nitrogen oxides (NOJ (8,24,25). NOx biofilters have been developed using a consortia of denitrifying microbes that have the physiological capacity, as a part of their normal metabolism, to use a variety of NOx compounds as terminal electron acceptors, reducing them to innocuous nitrogen gas (N2). This process generally requires low-oxygen conditions because denitrifiers preferentially use oxygen, if present, as their terminal electron acceptor. As a result, selective conditions for denitrifying microbes include a suitable carbon and energy source with a NOx compound as a terminal electron acceptor, a lack of oxygen, and the presence of supplemental nutrients necessary for microbial growth. The potential application to stack gases raises the question of the maximum temperature at which the microbes will function. Research (25) has shown that naturally occurring thermophilic microbes selected for from compost are capable of reducing nitric oxide (NO) to nitrogen gas (N2) at temperatures up to 60 °C.

Other processes are far more sensitive than denitrifi-cation to trace amounts of oxygen. The sulfite-reducing bacteria responsible for the removal of SO2 are obligate anaerobes, although they may be active in anoxic microniches, for example, deep in a biofilm. Chlorinated aliphatic solvents will also accept electrons under anaerobic conditions, a degradative mechanism that works best on the completely chlorinated compounds that are not touched by the oxidative enzymes already discussed (22,23). Carbon tetrachloride, for example, will be progressively reduced to chloroform, dichloromethane, and methyl chloride. This mechanism is often categorized as cometab-olism, implying that the microbes derive no metabolic benefit from it, but this remains uncertain.

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