RESEARCH NEEDS FOR BIOAUGMENTATION - Bioaugmentation for Groundwater Remediation - page 329

to a variety of cultures and will assist in assigning function to many dehalogenases only known
from a gene sequence.
Efforts also are currently directed at elucidating all proteins in the electron transport
chain (e.g., hydrogenases, cytochromes, ferredoxin, or other electron carriers), the mechanism
of reductive dehalogenation, its regulation and the specific conditions under which the different
dehalogenases are recruited. Similar interests exist for understanding the oxygenases and other
enzymes involved in aerobic mechanisms of dechlorination. The ever-increasing availability of
genomic data provides a mine for identifying genes encoding undiscovered biocatalysts for
biotransformation reactions. However, much work remains to fully characterize many of the
poorly annotated genes and proteins whose activities are correlated with biotransformation.

Research Needs: Basic Science - Molecular Scale
Mechanistic understanding of the reactions that break down pollutants in groundwater.

Identification of substrates and molecular mechanisms of enzyme-catalyzed contaminant
transformation reactions in biodegrading microbial communities.

12.2.2 Organismal Scale

A variety of aerobic and anaerobic bacteria capable of contaminant transformation
have been isolated in pure culture. Pure cultures derived from a single cell (i.e., clones) provide
a clean system in which to test hypotheses and understand the specific growth requirements of an
organism. Unfortunately, cultivated strains drastically under-represent the microbial
and functional diversity in the environment, in microcosms, enrichment cultures, and bio-
augmentation cultures. Novel cultivation techniques are needed not only to increase the number
of isolated strains, but also to study defined microbial communities in action. Certainly genome
and metagenome sequences and genome-wide assays (microarrays and shotgun proteomics) have
been very useful in helping to identify gene and protein responses to specific stresses and growth
conditions (N'guessan et al., 2010 ; Nicolau et al., 2009 ;Selesietal., 2010 ;Zhou, 2003 ).
Better understanding of an organism's nutrient requirements through identification of up-
regulated transport and synthesis systems may provide clues to better cultivate these organ-
isms, with the potential for increased yields and contaminant degradation rates. In particular,
genome-scale mathematical models of microbial metabolism and regulatory networks, which
provide a framework onto which to anchor disparate “omic” data, are a particularly powerful
tool for extracting useful information from studies of uncultivated organisms (Lee et al., 2006 ;
Zhao et al., 2010 ).
Despite the advantages of the novel “omic” technologies that can examine a mixed
bioaugmentation culture as a whole, highly enriched or purified strains continue to be the
basic unit for building the tree of life, and understanding cellular evolution. A specific example
of the benefits of prospecting for and isolating new organisms is provided by the search for
Chloroflexi (Section 2.2.1 ) . Genome-enabled models of microorganisms and their impact are
described in Section 2.2.2 . Both of these approaches have the potential to impact bioremediation
and bioaugmentation strategies.

12.2.2.1 Expanding the Dechlorinators: Novel Chloroflexi and Beyond
The discovery of Dehalococcoides strain 195 as a dechlorinating organism (Freedman
and Gossett, 1989 ; Maym ´ -Gatell et al., 1997 ) marked the beginning of enquiry into this
group as potential bioremediation tools. Members of the Dehalococcoides group have been