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   Anaerobic Microbiological Characterizations


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CytoCulture has added new microbiological assays for monitoring in situ bioremediation sites. This work is carried out in our new anaerobic glovebox under a 10%CO2 5%H2 85N2 atmosphere.

Anaerobic Site Characterizations

Anaerobic microbial activity is carried out in the absence of O2 as a terminal electron acceptor (TEA) in respiration. Alternatives to oxygen in respiration may be N03-, FeIII, MnIV, SO42- , and even CO2 in the case of methanogenesis.

Depending upon the availability of these TEAs and of carbon sources, anaerobic microorganisms will often set up "conditions" of microbial activity based upon the most energetically favorable TEA. For example, if sufficient NO3- is present in the absence of O2, the environment is said to be nitrate reducing. Likewise, conditions may also exist for an iron reducing, sulfate reducing, or methanogenic environment. A particular site may have zones with one or more of these conditions.

This is of particular relevance to biodegradation since research has shown that different organic contaminants, such as benzene, toluene, and chlorinated compounds will have different microbial degradation rates depending upon these TEA conditions. Rates under nitrate reducing conditions are often faster than under methanogenic conditions as nitrate reduction is more energetically favorable. Similarly, aromatic compounds may biodegrade more readily under nitrate reducing conditions than under sulfate reducing conditions. Some compounds, such as chlorinated compounds and MTBE, may actually biodegrade at higher rates under the proper anaerobic conditions than under aerobic conditions.

Therefore, it is critical that an understanding of the anaerobic site conditions be gained before an assessment of bioremediation potential activity can be made. General biodegradation predictions can be made from an introductory site characterization, and realistic microcosm studies can then be set up based upon this information.

Recommended site characterization protocol:

1) A characterization of the presence of the major physiological groups of anaerobes using MPNs and plate counting from the site. We would test for the presence of nitrate, iron, and sulfate reducing bacteria using our new anaerobic MPN enumeration services. We would also test for the presence of total heterotrophic bacteria and hydrocarbon degrading bacteria (anaerobic and aerobic) with our own plate count enumeration protocols.

2) A concurrent characterization of the inorganic chemistry of the site will also provide data on subsurface conditions able to support anaerobic biodegradation of target contaminants. CytoCulture would perform the following tests at our laboratory: Sulfate, Nitrate-N, Ammonia-N, Ortho-Phosphate, Dissolved Oxygen, redox potential (mV), and pH. We would subcontract testing for ferrous and ferric iron, sulfide, and methane.

Anaerobic plate counts for hydrocarbon-degraders and total heterotrophs

These assays are similar in principle to our aerobic assays, except that they are performed in the absence of oxygen. Alternate electron acceptors such as sulfate, nitrate, and ferric iron are added to the media to meet anaerobic respiration needs. A standard anaerobic agar (DIFCO) is used for total anaerobic heterotrophic plate counts. For anaerobic hydrocarbon degraders, a combination of diesel and jet fuel is added to the media as sole carbon sources. A minimal salts mixture and trace elements are added to meet growth requirements.

Bacteria enumerations by MPN method for anaerobic iron reducers, nitrate reducers and sulfate reducers

Our most probable number (MPN) techniques are adaptations of the classical Standard Methods technique originally developed for the enumeration of Coliform bacteria in wastewater. Specialized media are used for each anaerobic group tested for.


MPN Methods Used to Determine Anaerobes
Physiological group Media used Growth indicator Reference
Fe(III) Reducers FWA medium with ferric citrate as TEA and acetate as sole carbon source. Color change:

brownÞ greenÞ clear

Lovley et al. 1988
Nitrate Reducers Nitrate Broth Bray’s Nitrate Powder

Color Change

Focht and Joseph, 1973
Sulfate Reducers SR medium B FeS black precipitate. Postgate et al.., 1979


Anaerobic microcosm studies to determine biodegradability/ biodegradability potential

Although biodegradation rates can often be inferred by a combination of microbial site characterization data and historical contaminant chemistry data, laboratory biodegradation microcosm studies can often be an effective tool in determining contaminant breakdown rates. These studies follow the generalized protocol below:

  • Using site characterization data, microcosms are designed to mimic the anaerobic site conditions (nitrate-, sulfate-, iron- reducing, or methanogenic).
  • Using soil, sediment, and/ or water from the site containing indigenous microbes, microcosms are set up as serum bottle (water or slurry) or flow-through column (soil or sediment) in the anaerobic glovebox.
  • The desired contaminant(s) are added.
  • Changes in contaminant concentrations through time are monitored to establish breakdown rates.

Microcosm studies can be designed in which microbial parameters can be measured throughout the assay, or in which specific breakdown products can be quantified.

See also: Monitored Natural Attenuation (MNA) info


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