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
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
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
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
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.